Methods for quantifying polypeptides using mass spectrometry

ABSTRACT

A method for identifying a polypeptide a specimen can include (i) treating a specimen suspected of including an insulin with a base; (ii) extracting a first fraction of the treated specimen by solid phase extraction using a mixed mode or polymeric reversed-phase media and a first solvent including an acid; (iii) separating a component of the first fraction by liquid chromatography using a chromatographic surface including a hydrophobic surface group and one or more ionizable modifiers, and a second solvent including an acid; and (iv) analyzing the component of the first fraction by mass spectroscopy, thereby identifying the polypeptide, if present, using a signal corresponding to a sequence fragment ion from the polypeptide. The signal can correspond to an intact multiply charged precursor fragment selected in a first quadrupole and its corresponding sequence fragment ion selected in a final quadrupole.

RELATED APPLICATIONS

This application is a continuation of U.S. utility application Ser. No.15/447,838, filed on Mar. 2, 2017, and will issue as U.S. Pat. No.9,964,545 on May 8, 2018. U.S. utility application Ser. No. 15/447,838is a continuation of U.S. utility application Ser. No. 14/394,952, filedon Oct. 16, 2014, and issued as U.S. Pat. No. 9,588,130 on Mar. 7, 2017.U.S. utility application Ser. No. 14/394,952 is a National StageApplication of International Application No. PCT/US2013/031595, filedMar. 14, 2013, which claims priority to U.S. Provisional ApplicationNos. 61/635,013, filing date Apr. 18, 2012; 61/649,404, filing date May21, 2012; and 61/740,973, filing date Dec. 21, 2012. Each of theforegoing applications is, incorporated herein by reference in itsentirety.

FIELD OF THE TECHNOLOGY

The technology relates generally to methods for quantifying one or morepolypeptides in a sample by mass spectrometry. The technology relatesmore particularly, in various embodiments, to combinations of mixed-modeor reversed-phase solid phase extraction, liquid chromatographyincluding use of a chromatographic surface having a hydrophobic surfacegroup and one or more ionizable modifiers, and high sensitivity massspectroscopy for quantification of polypeptides.

BACKGROUND OF THE TECHNOLOGY

Polypeptides are commonly analyzed using ligand binding assays (LBA)such as enzyme-linked immunosorbent assays (ELISA). However, ELISA canlack specificity and accuracy, for example due to cross reactivity andan inability to distinguish similar molecules such as analogs andmetabolites. Use of ligand binding assays such as ELISA for analyzingpolypeptides is also hindered when suitable antibodies are not yetavailable for the polypeptide of interest. Development of antibodies canbe an expensive and time-consuming process.

Although biologics have historically been quantified using ligandbinding assays (LBAs), over the past few years, there has been a trendtoward the analysis of large molecules by liquid chromatography tandemmass spectroscopy (LC-MS/MS). However, intact polypeptides such asinsulin are particularly difficult to analyze by LC-MS/MS. For example,mass spectroscopy (MS) sensitivity can be low due to poor transfer intothe gas phase and poor fragmentation due to the presence of multiplestabilizing disulfide bonds. In addition, polypeptides can suffer fromnon-specific binding and poor solubility, making liquid chromatography(LC) and sample preparation method development difficult.

SUMMARY OF THE TECHNOLOGY

In various aspects and embodiments, the technology includes methods forquantifying polypeptides by mass spectrometry, as well as correspondingcompositions, kits, apparatuses, and the like. For example, thetechnology includes combinations of multi-step mixed-mode solid phaseextraction, liquid chromatography column chemistry, wherein thechromatographic surface includes a hydrophobic surface group and one ormore ionizable modifiers (e.g. a charged surface hybrid column, forinstance an ACQUITY UPLC® CSH or XSelect™ column, commercially availablefrom Waters Technology Corp., Milford, Mass.) and high sensitivity massspectroscopy for quantification of polypeptides. The technology hasnumerous applications including polypeptide analysis, clinicaldiagnostics, medicine, biomarker and drug screening and discovery,bioequivalence testing, therapeutic monitoring, as well as relatedmethods in food, industrial, and environmental fields.

For polypeptide analytes (e.g., insulins, which are heavily stabilizedby disulfide bonds), it can be difficult to produce selective MSfragments because slight changes in collision energy result in littlefragmentation, rapidly followed by extensive fragmentation into verysmall, non-specific fragments. The technology is well suited forstudying such polypeptides. The technology is also generally applicableto quantifying large, low abundance peptides, mitigating non-specificbinding, and/or mitigating evaporative losses in the analysis ofpolypeptides.

One advantageous feature of the technology addresses these issues byusing a high sensitivity mass spectroscopy platform to produce specificpolypeptide fragments. For example, triple quadrupole MS can be used toproduce high m/z fragments that are analyte-specific (e.g., ranging fromm/z 700-1400 in the example of insulin). Accordingly, the technology canprovide a distinct selectivity advantage, reducing endogenousbackground, relative to use of lower m/z intense immonium ion fragments.

Another advantageous feature of the technology includes the use of achromatography column including a chromatographic surface wherein thechromatographic surface includes a hydrophobic surface group and one ormore ionizable modifiers, which can reduce secondary interactions andmimics the peak shape benefit for peptides that was historicallyachieved through the use of trifluoroacetic acid (TFA) in reversed-phasechromatography systems. Accordingly, the technology can obtain peakwidths that are significantly narrower than traditional columns usingformic acid in the mobile phase.

Yet another advantageous feature of the technology includes thesynergistic effect of combining a highly selective sample preparationmethod (e.g., multi-step mixed-mode or reversed phase polymeric SPE),coupled to a high resolution chromatographic method (e.g., LC columnchemistry wherein the chromatographic surface includes a hydrophobicsurface group and one or more ionizable modifiers), and a highsensitivity MS platform (e.g., triple quadrupole MS, analyte-specifichigh m/z fragments) for analyzing polypeptides. As a result, thetechnology is highly selective (e.g., can resolve related compounds thatELISA and MS methods relying on smaller/immonium ions cannot) and highlysensitive (e.g., effective in small samples and low concentrationsolutions) for large molecules such as polypeptides.

In one aspect, the technology features a method for identifying and/orquantifying a polypeptide in a specimen. The method includes (i)treating a specimen suspected of including a polypeptide with a base,(ii) extracting a first fraction of the treated specimen by solid phaseextraction using a mixed mode or polymeric reversed-phase media and afirst solvent including an acid, (iii) separating a component of thefirst fraction by liquid chromatography using a chromatographic surfaceincluding a hydrophobic surface group, and one or more ionizablemodifiers, and a second solvent including an acid, and (iv) analyzingthe component of the first fraction by mass spectroscopy, therebyidentifying and/or quantifying the polypeptide, if present, using asignal corresponding to a transition from a multiply charged precursorto a sequence fragment ion from the polypeptide.

In another aspect, the technology features a method for identifying aninsulin in a specimen. The method includes (i) treating a specimensuspected of including an insulin with a base, (ii) extracting a firstfraction of the treated specimen by solid phase extraction using a mixedmode or polymeric reversed-phase mode media and a first solventincluding an acid, (iii) separating a component of the first fraction byliquid chromatography using a chromatographic surface including ahydrophobic surface group and one or more ionizable modifiers, and asecond solvent including an acid, and (iv) analyzing the component ofthe first fraction by mass spectroscopy, thereby identifying theinsulin, if present, using a signal corresponding to a transition from amultiply charged precursor to a sequence fragment ion from the insulin.

In yet another aspect, the technology features a method for quantifyingan insulin (or other polypeptide) in a specimen. The method includes (i)treating a specimen suspected of including an insulin with2-amino-2-hydroxymethyl-propane-1,3-diol, (ii) extracting a firstfraction of the treated specimen by solid phase extraction using a mixedmode media operating in cation exchange and reverse phase modes and afirst solvent including acetic acid, (iii) separating a component of thefirst fraction by liquid chromatography using a chromatographic surfaceincluding a hydrophobic surface group and one or more ionizablemodifiers, and a second solvent including formic acid, and (iv)analyzing the component of the first fraction by triple quadrupole massspectroscopy operated in positive electrospray ionization mode, therebyquantifying the insulin, if present, using a signal corresponding to anintact multiply charged precursor fragment selected in a firstquadrupole and its corresponding sequence fragment ion selected in afinal quadrupole.

In still yet another aspect, the technology features a method forassessing the bioequivalence of a first polypeptide and a secondpolypeptide. The method includes (i) obtaining a specimen from abioequivalence assay for a first polypeptide, (ii) treating the specimenwith a base, (iii) extracting a first fraction of the treated specimenby solid phase extraction using a mixed mode or polymeric reversed-phasemedia and a first solvent including an acid, (iv) separating a componentof the first fraction by liquid chromatography using a chromatographicsurface including a hydrophobic surface group and one or more ionizablemodifiers, and a second solvent including an acid, (v) analyzing thecomponent of the first fraction by mass spectroscopy, therebyquantifying the first polypeptide using a signal corresponding to asequence fragment ion from the first polypeptide, and (vi) comparing thequantity of the first polypeptide to a quantity of the secondpolypeptide expected in a bioequivalence assay for a first polypeptide,thereby determining if the first polypeptide and second polypeptide arebioequivalent.

Any of the above aspects can be combined with any one or more of theembodiments listed below and/or any one or more features provided in thespecification and drawings.

In various embodiments, a method can further include quantifying theamount of the insulin (or other polypeptide), if present, using thesignal corresponding to the sequence fragment ion from the insulin,where the signal corresponds to an intact multiply charged precursorfragment selected in a first quadrupole and its corresponding sequencefragment ion selected in a final quadrupole, and where the sequencefragment ion exhibits an m/z>800. Where the method is used on anotherpolypeptide, the sequence fragment ion can exhibit an m/z that ischaracteristic of that polypeptide in an undigested state.

In some embodiments, the base includes2-amino-2-hydroxymethyl-propane-1,3-diol.

In certain embodiments, treating the specimen further includes anorganic precipitation.

In various embodiments, the mixed mode media includes ion exchangemoieties and reverse phase moieties. The ion exchange can include cationexchange.

In some embodiments, the first solvent includes acetic acid. The secondsolvent can include acetic acid and/or formic acid.

In certain embodiments, the component of the first fraction includesundigested insulin. Where the method is used on another polypeptide, thefirst fraction can include that polypeptide in an undigested state.

In various embodiments, the mass spectrometry includes triple quadrupolemass spectrometry. The mass spectrometry can be carried out in positiveelectrospray ionization mode.

In some embodiments, the sequence fragment ion exhibits an m/z>800.Where the method is used on another polypeptide, the sequence fragmention can exhibit an m/z that is characteristic of that polypeptide in anundigested state.

In certain embodiments, the detection limit of the insulin (orpolypeptide of interest) is 0.25 ng/mL or less. The detection limit ofthe polypeptide of interest can be 0.50 ng/mL or less. The detectionlimit can be achieved in a specimen of 250 microliters or less. Incertain embodiments, the detection limit for a polypeptide of interestcan be 30 pg/mL. In some embodiments, the detection limit for apolypeptide of interest can be 15 pg/mL.

In various embodiments, analyzing the component of the first fraction bymass spectroscopy can resolve any two or more of insulin glargine,insulin detemir, insulin aspart, insulin glulisine, or human insulin.

In some embodiments, the method further includes quantifying the amountof the insulin, if present, using the signal corresponding to thesequence fragment ion from the insulin.

In certain embodiments, the signal corresponds to an intact multiplycharged precursor fragment selected in a first quadrupole and itscorresponding sequence fragment ion selected in a final quadrupole.

In various embodiments, bioequivalence is determined based upon theabsence of a significant difference in the rate and extent to which thefirst polypeptide (e.g., first insulin) and second polypeptide (e.g.,second insulin) become available at the site of drug action whenadministered at the same molar dose under similar conditions in thebioequivalence assay.

In some embodiments, the bioequivalence assay includes a pharmacokineticstudy.

In certain embodiments, the bioequivalence assay includes apharmacodynamics study.

In various embodiments, the bioequivalence assay includes a clinicaltrial.

In some embodiments, the bioequivalence assay includes an in vitro test.

In certain embodiments, the first polypeptide and the second polypeptideare each independently insulin glargine, insulin detemir, insulinaspart, insulin glulisine, human insulin, or a derivative or analogthereof.

In various embodiments, the first polypeptide and the second polypeptideare each independently exenatide, hepcidin, teriparatide, enfuvirtide,calcitonin, brain natriuretic peptide (BNP), amyloid beta peptides,GLP-1, glucagon, bombesin, or a derivative or analog thereof.

In some embodiments, analyzing the component of the first fraction bymass spectroscopy can resolve any two or more of insulin glargine,insulin detemir, insulin aspart, insulin glulisine, human insulin, or aderivative or analog thereof.

In certain embodiments, analyzing the component of the first fraction bymass spectroscopy can resolve any two or more of exenatide, hepcidin,teriparatide, enfuvirtide, calcitonin, brain natriuretic peptide (BNP),amyloid beta peptides, GLP-1, glucagon, bombesin, or a derivative oranalog thereof.

The present technology is described in further detail by the figures andexamples below, which are used only for illustration purposes and arenot limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a MS scan of Lantus® (insulin glargine).

FIG. 2 shows a MS/MS scan of m/z 867 for Lantus® (insulin glargine).

FIG. 3 shows a chromatogram for the transition monitoring of Lantus®(insulin glargine).

FIGS. 4A-4C shows chromatograms for Lantus® (insulin glargine) atvarious concentrations. FIGS. 4D-4F shows integrated analyte peaks fromthe chromatograms shown in FIGS. 4A-4C.

FIGS. 5A-5C shows the linearity for Lantus® (insulin glargine) insolvent standards.

FIGS. 6A-6C shows chromatograms for Lantus® (insulin glargine) extractedfrom human plasma and analyzed in accordance with the technology.

FIGS. 7A-7E shows chromatograms for additional levels and integrationfor Lantus® (insulin glargine).

FIG. 8A shows a MS scan of Levemir® (insulin detemir). FIG. 8B shows aMS/MS scan of m/z 1184 for Levemir® (insulin detemir).

FIGS. 9A-9D shows chromatograms for Levemir® (insulin detemir) atvarious concentrations. FIGS. 9E-9F shows an integrated analyte peakfrom the chromatograms shown in FIGS. 9A-9D.

FIGS. 10A-10C shows the linearity for Levemir® (insulin detemir) insolvent standards.

FIGS. 11A-11C shows chromatograms and peak integrations for Levemir®(insulin detemir) extracted from human plasma and analyzed in accordancewith the technology.

FIGS. 12A-12C shows chromatograms and peak integrations for Levemir®(insulin detemir) extracted from human plasma and analyzed in accordancewith the technology.

FIG. 13 shows a MS scan of Apidra® (insulin glulisine).

FIGS. 14A-14C shows chromatograms for Apidra® (insulin glulisine) atvarious concentrations.

FIGS. 15A-15C shows the linearity for Apidra® (insulin glulisine) insolvent standards.

FIGS. 16A-16C shows chromatograms and peak integrations for Apidra®(insulin glulisine) extracted from human plasma and analyzed inaccordance with the technology.

FIG. 17 shows a MS scan of Novolog/Novorapid® (insulin aspart).

FIGS. 18A-18D shows chromatograms for Novolog/Novorapid® (insulinaspart) at various concentrations. FIGS. 18E-18F shows an integratedanalyte peak from the chromatograms shown in FIGS. 18A-18D.

FIGS. 19A-19C shows the linearity for Novolog/Novorapid® (insulinaspart) in solvent standards.

FIGS. 20A-20C shows chromatograms for injections for Novolog/Novorapid®(insulin aspart) extracted from human plasma and analyzed in accordancewith the technology.

FIGS. 21A-21B shows example chromatograms from final eluates fromsamples pretreated with TRIS.

FIGS. 21C-21D shows example chromatograms from final eluates fromsamples pretreated with TFA.

FIGS. 22A-22D shows representative MSMS spectra for insulin analogs.

FIGS. 23A-23C show representative chromatograms of an extracted plasmablank and insulin glulisine at the LOD and LLOQ.

DETAILED DESCRIPTION OF THE TECHNOLOGY

The technology provides methods, as well as corresponding composition,kits, and apparatuses for quantifying a polypeptide analyte in a sample.The technology can employ combinations of mixed-mode or reversed phasesolid phase extraction, liquid chromatography using a chromatographicsurface including a hydrophobic surface group and one or more ionizablemodifiers, (e.g., with columns having <2 micrometer particles), and highsensitivity mass spectroscopy for identification and quantification ofpolypeptides. MS can employ sequence fragment ions from the polypeptide,for example, intact multiply charged precursor fragments selected in afirst quadrupole and corresponding sequence fragment ion selected in afinal quadrupole, where the sequence fragment ion exhibits an m/z thatis characteristic of that polypeptide in an undigested state. An examplechromatographic surface including a hydrophobic surface group and one ormore ionizable modifiers can be a charged surface hybrid column, forinstance an ACQUITY UPLC® CSH or XSelect™ column, commercially availablefrom Waters Technology Corp., Milford, Mass. The following detaileddescription provides additional description of the analytes and samples,followed by the pre-treatment, separation, and analysis steps and,finally, illustrative examples.

Analytes

Further to the summary above, analytes or target polypeptide analytescan include essentially any polypeptide of interest that can be detectedusing a mass spectrometer. The target analyte can be of interest, forexample, in one or more of clinical chemistry, medicine, veterinarymedicine, forensic chemistry, pharmacology, food industry, safety atwork, and environmental pollution.

Clinical chemistry target analytes can include any polypeptide presentin an organism (e.g., human body, animal body, fungi, bacterium, virus,and the like). For example, clinical chemistry target analytes include,but are not limited to, proteins, protein metabolites, proteinbiomarkers, and polypeptide drugs and their metabolites.

Human medicine and veterinary medicine target analytes can include anypolypeptide that can be used for the diagnosis, prophylaxis or treatmentof a disease or condition in a subject. For example, human medicine andveterinary medicine target analytes include, but are not limited to,disease markers, prophylactic, or therapeutic agents.

Forensic chemistry target analytes can include any polypeptide presentin a sample taken from the site of crime, such as a sample from avictim's body (e.g., tissue or fluid sample, hair, blood, semen, urine,and the like). For example, clinical chemistry target analytes include,but are not limited to, toxic agents, drugs and their metabolites,biomarkers, and identifying compounds.

Pharmacology target analytes can include any polypeptide that is apharmaceutical or metabolite thereof or which can be used for thedesign, synthesis, and monitoring of drugs. For example, pharmacologytarget analytes include, but are not limited to, polypeptideprophylactic and/or therapeutic agents, their prodrugs, intermediatesand metabolites. Pharmacological analysis can include bioequivalencetesting, for example, in connection with the approval, manufacturing,and monitoring of a generic drug.

Food industry and agricultural target analytes can include anypolypeptide that is relevant for monitoring of the safety of foods,beverages, and/or other food industry/agricultural products. Examples oftarget analytes from the field of food industry include, but are notlimited to, polypeptide pathogen markers, allergens (e.g., gluten andnut proteins), and mycotoxins.

Target analytes can include polypeptides (e.g., polymers of naturallyand/or non-naturally occurring amino acids such as Gly, Ala, Val, Leu,Ile, Pro, Phe, Trp, Cys, Met, Ser, Thr, Tyr, His, Lys, Arg, Asp, Glu,Asn, Gln, selenocysteine, ornithine, citrulline, hydroxyproline,methyllysine, carboxyglutamate), peptides, polypeptides, proteins,glycoproteins, lipoproteins; peptide-nucleic acids; hormones (such aspeptide hormones (e.g., TRH and vasopressin), as well as synthetic andindustrial polypeptides.

In some embodiments, target analytes can include peptides and/orpolypeptides that are difficult to identify and/or quantify byconventional methods (e.g., ligand binding assays such as ELISA). Suchpeptides or polypeptides can be difficult to identify and/or quantifybecause no antibody to the polypeptide of interest is available, and/orbecause it is difficult to distinguish the polypeptide analyte from ametabolite or analog thereof.

Furthermore, in some embodiments, target peptides or polypeptides can bedifficult to analyze by conventional methods (e.g., ligand bindingassays such as ELISA) because they exhibit a high degree of non-specificbinding, they are greater than about 3,000 Daltons in molecular weight,they form aggregates, they fragment poorly in a mass spectrometer, theyare highly polar (e.g., they require mixed-mode SPE approaches in orderto efficiently bind and release them during sample preparation), theyare highly non-polar (e.g., they require a high percentage of modifiersto release them from extraction media and keep them in solution), theyneed to be separated from isobaric peptides in the sample that are atmuch higher concentrations, they have poor transfer efficiency in andout of chromatographic pools of LC and SPE columns, they suffer fromsignificant secondary interactions with chromatographic media resultingin poor peak shape and/or inability to elute in a highly resolved band,they are unstable, and/or they undergo chemical modification underextreme pH conditions.

In some embodiments, target analyte peptides or polypeptides can includeone or more of exenatide, hepcidin, teriparatide, enfuvirtide,calcitonin, brain natriuretic peptide (BNP), amyloid beta peptides,e.g., GLP-1, glucagon, bombesin, and derivatives or analogs thereof, andthe like. In some embodiments, the target analyte can include one ormore insulin, and/or insulin analogs such as Lantus® (insulin glargine),Levemir® (insulin detemir), Novolog/NovoRapid® (insulin aspart), andApidra® (insulin glulisine).

Samples

In general, a sample is a composition including at least one targetanalyte (e.g., an analyte of the class or kind disclosed above, togetherwith a matrix). Samples can include a solid, liquid, gas, mixture,material (e.g., of intermediary consistency, such as an extract, cell,tissue, organisms) or a combination thereof. In various embodiments, thesample is a bodily sample, an environmental sample, a food sample, asynthetic sample, an extract (e.g., obtained by separation techniques),or a combination thereof.

Bodily samples can include any sample that is derived from the body ofan individual. In this context, the individual can be an animal, forexample a mammal, for example a human. Other example individuals includea mouse, rat, guinea-pig, rabbit, cat, dog, goat, sheep, pig, cow, orhorse. The individual can be a patient, for example, an individualsuffering from a disease or being suspected of suffering from a disease.A bodily sample can be a bodily fluid or tissue, for example taken forthe purpose of a scientific or medical test, such as for studying ordiagnosing a disease (e.g., by detecting and/or identifying a pathogenor the presence of a biomarker). Bodily samples can also include cells,for example, pathogens or cells of the individual bodily sample (e.g.,tumor cells). Such bodily samples can be obtained by known methodsincluding tissue biopsy (e.g., punch biopsy) and by taking blood,bronchial aspirate, sputum, urine, feces, or other body fluids.Exemplary bodily samples include humor, whole blood, plasma, serum,umbilical cord blood (in particular, blood obtained by percutaneousumbilical cord blood sampling (PUBS), cerebrospinal fluid (CSF), saliva,amniotic fluid, breast milk, secretion, ichor, urine, feces, meconium,skin, nail, hair, umbilicus, gastric contents, placenta, bone marrow,peripheral blood lymphocytes (PBL), and solid organ tissue extract.

Environmental samples can include any sample that is derived from theenvironment, such as the natural environment (e.g., seas, soils, air,and flora) or the manmade environment (e.g., canals, tunnels,buildings). Exemplary environmental samples include water (e.g.,drinking water, river water, surface water, ground water, potable water,sewage, effluent, wastewater, or leachate), soil, air, sediment, biota(e.g., soil biota), flora, fauna (e.g., fish), and earth mass (e.g.,excavated material).

Food samples can include any sample that is derived from food (includingbeverages). Such food samples can be used for various purposesincluding, for example, (1) to check whether a food is safe; (2) tocheck whether a food contained harmful contaminants at the time the foodwas eaten (retained samples) or whether a food does not contain harmfulcontaminants; (3) to check whether a food contains only permittedadditives (e.g., regulatory compliance); (4) to check whether itcontains the correct levels of mandatory ingredients (e.g., whether thedeclarations on the label of the food are correct); or (5) to analyzethe amounts of nutrients contained in the food. Exemplary food samplesinclude edible products of animal, vegetable or synthetic origin (e.g.,milk, bread, eggs, or meat), meals, drinks, and parts thereof, such asretain samples. Food samples can also include fruits, vegetables,pulses, nuts, oil seeds, oil fruits, cereals, tea, coffee, herbalinfusions, cocoa, hops, herbs, spices, sugar plants, meat, fat, kidney,liver, offal, milk, eggs, honey, fish, and beverages.

Synthetic samples can include any sample that is derived from anindustrial process. The industrial process can be a biologicalindustrial process (e.g., processes using biological material containinggenetic information and capable of reproducing itself or beingreproduced in a biological system, such as fermentation processes usingtransfected cells) or a non-biological industrial process (e.g., thechemical synthesis or degradation of a compound such as apharmaceutical). Synthetic samples can be used to check and monitor theprogress of the industrial process, to determine the yield of thedesired product, and/or measure the amount of side products and/orstarting materials.

Pre-Treatment

Further to the summary above, the technology includes (i) treating aspecimen suspected of including a polypeptide with a base and (ii)extracting a first fraction of the treated specimen by solid phaseextraction using a mixed mode or a polymeric reversed-phase media and afirst solvent including an acid. The pre-treatment steps of thetechnology work in concert with the separation and analysis steps, toprovide a high resolution tool for studying polypeptides. In variousembodiments, the pre-treatment can be adapted to avoid, minimize,mitigate, or otherwise control the digestion of the polypeptide analyteand to impart further selectivity onto the final extract.

Treatment with a base can include mixing the sample with bases used inthe art for pre-treatment (e.g., as opposed to prior art methods thatcall for dilution of the sample with an acid). For example, thetreatment can use TRIS base (e.g.,2-amino-2-hydroxymethyl-propane-1,3-diol). Treatment with base can becarried out prior to loading the sample for extraction. Treatment withbase can eliminate potentially interfering polypeptides such as serumalbumin from the sample, thereby improving the quality of the analysis.

Extracting a first fraction of the treated specimen by solid phaseextraction (SPE) can include using a mixed mode or a polymericreversed-phase media and a first solvent including an acid. In variousembodiments, the mixed mode media includes ion exchange moieties andreverse phase moieties. The ion exchange can include cation exchange. Anexample SPE media is a Waters Oasis® HLB μElution 96-well plate.

Accordingly, the extraction (e.g., in combination with the separation)imparts orthogonality into the methods of the technology, by separatingon the basis of different physical properties (e.g., both charge andhydrophobicity).

In various embodiments, elution solvents (e.g., in pre-treatment and/orseparation) include an acid. An example solvent is acetic acid inwater/organic. The acid in the solvent can facilitate solubilization ofthe polypeptide analyte and mitigate adsobtion of the polypeptideanalyte during processing. The use of acid allows for the minimizationor elimination of organic solvents, which can cause polypeptideprecipitation.

In certain embodiments, the technology can include one or moreadditional pre-treatment steps or techniques. For example, treating thespecimen can also include an organic precipitation.

In certain embodiments, dilution with TRIS base at pH 9-9.5 instead ofdilution with an acid significantly improved recovery and reducedendogenous background. For instance, FIGS. 21A and 21C showschromatograms from the final eluates from samples pretreated with TFAcontained a broad, intense interference peak at 5.76 minutes that wasabsent from eluates pretreated with TRIS. These samples were furtheranalyzed using full scan MS to elucidate the nature of the peak. Spectrawere summed from 5.5 to 6.25 minutes samples from both pre-treatments,and the resultant data are shown in the bottom panels of FIGS. 21B and21D. Deconvolution of the protein envelope in the samples pretreatedwith TFA produced an intact MW of approximately 66,400, providingputative identification as human serum albumin (HSA). HSA is typicallypresent at approximately 35-50 mg/mL and must be efficiently removed andseparated from the insulin analytes. Pretreatment with TRIS efficientlyachieved this goal, as evidenced by the absence of the large proteinpeak at 5.66 minutes.

Separation

Further to the summary above, the technology includes separating acomponent of the first fraction by liquid chromatography using achromatographic surface including a hydrophobic group surface and one ormore ionizable modifiers, and a second solvent including an acid. Theseparation step of the technology works in concert with thepre-treatment and analysis steps, to provide a high resolution tool forstudying polypeptides. In various embodiments, the separation can beadapted to avoid, minimize, mitigate, or otherwise control the digestionof the polypeptide analyte.

The chromatography columns of the technology can include achromatographic surface having a hydrophobic surface group and one ormore ionizable modifiers, and can facilitate separations by minimizingsecondary interactions (e.g., due to + charges on the stationary phasesurface). The chromatography columns can also allow for the use offormic acid in the mobile phase (e.g., in contrast to the TFA in themobile phase). Accordingly, the technology provides for narrow peaks(e.g., 2-4× narrower than conventional chromatography setups), whichimproves signal to noise ratios and detection limits (e.g., especiallyfor analytes at low concentration). One example chromatographic surfaceincluding a hydrophobic surface group and one or more ionizablemodifiers is a Waters ACQUITY CSH™ C18 2.1×50 mm, 1.7 μm column. Thesecond solvent can include formic acid (e.g., 0.1-1% formic acid inwater).

In some embodiments, addition of a carrier protein can be an effectiveway to minimize nonspecific binding (NSB). For instance, for simplicity,0.05% rat plasma can be added to the sample diluents, resulting inimproved linearity in solvent standards. NSB can also occur betweenpeptide analytes and the chromatographic columns LC columns may have tobe “pretreated” by injection of protein precipitated plasma in order toobtain the best performance for biomolecules such as insulin. The plasmacomponents presumably coat the column surface and effectively minimizeNSB. In certain embodiments, these changes can result in reproduciblepeak areas and a broad linear dynamic range in solvent standards as wellas an easily achievable LLOQ of 50 pg/mL for solvent standards.

Chromatographic Surface Materials

Chromatographic surface materials for use with the instant invention caninclude high purity chromatographic materials (HPCMs). For example, achromatographic surface can incluse a hydrophobic surface group and oneor more ionizable modifiers. Chromatographic surfaces can be used, forexample, in chromatographic columns. In some embodiments, the ionizablemodifier does not contain a Zwitterion, the ionizable modifier does notcontain a quaternary ammonium ion moiety.

The term “high purity” or “high purity chromatographic material” caninclude a material which is prepared from high purity precursors. Incertain aspects, high purity materials have reduced metal contaminationand/or non-diminished chromatographic properties including, but notlimited to, the acidity of surface silanols and the heterogeneity of thesurface.

The term “chromatographic surface” can include a surface which providesfor chromatographic separation of a sample. In certain aspects, thechromatographic surface is porous. In some aspects, a chromatographicsurface may be the surface of a particle, a superficially porousmaterial or a monolith. In certain aspects, the chromatographic surfaceis composed of the surface of one or more particles, superficiallyporous materials or monoliths used in combination during achromatographic separation. In certain other aspects, thechromatographic surface is non-porous.

The term “ionizable modifier” can include a functional group which bearsan electron donating or electron withdrawing group. In certain aspects,the ionizable modifier contains one or more carboxylic acid groups,amino groups, imido groups, amido groups, pyridyl groups, imidazolylgroups, ureido groups, thionyl-ureido groups or aminosilane groups, or acombination thereof. In other aspects, the ionizable modifier contains agroup bearing a nitrogen or phosphorous atom having a free electron lonepair. In certain aspects, the ionizable modifier is covalently attachedto the material surface and has an ionizable group. In some instances itis a attached to the chromatographic material by chemical modificationof a surface hybrid group.

The term “hybrid”, including “hybrid inorganic/organic material,” caninclude inorganic-based structures wherein an organic functionality isintegral to both the internal or “skeletal” inorganic structure as wellas the hybrid material surface. The inorganic portion of the hybridmaterial may be, e.g., alumina, silica, titanium, cerium, or zirconiumor oxides thereof, or ceramic material. “Hybrid” includesinorganic-based structures wherein an organic functionality is integralto both the internal or “skeletal” inorganic structure as well as thehybrid material surface. As noted above, exemplary hybrid materials areshown in U.S. Pat. Nos. 4,017,528, 6,528,167, 6,686,035 and 7,175,913.

The term “hydrophobic surface group” can include a surface group on thechromatographic surface which exhibits hydrophobicity. In certainaspects, a hydrophobic group can be a carbon bonded phase such as a C4to C18 bonded phase. In other aspects, a hydrophobic surface group cancontain an embedded polar group such that the external portion of thehydrophobic surface maintains hydrophobicity. In some instances it is aattached to the chromatographic material by chemical modification of asurface hybrid group. In other instances the hydrophobic group can beC4-C30, embedded polar, chiral, phenylalkyl, or pentafluorophenylbonding and coatings.

In certain aspects the HPCM may further comprise a chromatographic corematerial. In some aspects, the chromatographic core is a silicamaterial; a hybrid inorganic/organic material; a superficially porousmaterial; or a superficially porous particle. The chromatographic corematerial may be in the form of discreet particles or may be a monolith.The chromatographic core material may be any porous material and may becommercially available or may be produced by known methods, such asthose methods described in, for example, in U.S. Pat. Nos. 4,017,528,6,528,167, 6,686,035 and 7,175,913. In some embodiments, thechromatographic core material may be a non-porous core.

The term “monolith” is intended to include a collection of individualparticles packed into a bed formation, in which the shape and morphologyof the individual particles are maintained. The particles areadvantageously packed using a material that binds the particlestogether. Any number of binding materials that are well known in the artcan be used such as, for example, linear or cross-linked polymers ofdivinylbenzene, methacrylate, urethanes, alkenes, alkynes, amines,amides, isocyanates, or epoxy groups, as well as condensation reactionsof organoalkoxysilanes, tetraalkoxysilanes, polyorganoalkoxysiloxanes,polyethoxysiloxanes, and ceramic precursors. In certain embodiments, theterm “monolith” also includes hybrid monoliths made by other methods,such as hybrid monoliths detailed in U.S. Pat. No. 7,250,214; hybridmonoliths prepared from the condensation of one or more monomers thatcontain 0-99 mole percent silica (e.g., SiO₂); hybrid monoliths preparedfrom coalesced porous inorganic/organic particles; hybrid monoliths thathave a chromatographically-enhancing pore geometry; hybrid monolithsthat do not have a chromatographically-enhancing pore geometry; hybridmonoliths that have ordered pore structure; hybrid monoliths that havenon-periodic pore structure; hybrid monoliths that have non-crystallineor amorphous molecular ordering; hybrid monoliths that have crystallinedomains or regions; hybrid monoliths with a variety of differentmacropore and mesopore properties; and hybrid monoliths in a variety ofdifferent aspect ratios. In certain embodiments, the term “monolith”also includes inorganic monoliths, such as those described in G.Guiochon/J. Chromatogr. A 1168 (2007) 101-168.

The term “chromatographic core” can include chromatographic materials,including but not limited to an organic material such as silica or ahybrid material, as defined herein, in the form of a particle, amonolith or another suitable structure which forms an internal portionof the materials of the invention. In certain aspects, the surface ofthe chromatographic core represents the chromatographic surface, asdefined herein, or represents a material encased by a chromatographicsurface, as defined herein. The chromatographic surface material may bedisposed on or bonded to or annealed to the chromatographic core in sucha way that a discrete or distinct transition is discernible or may bebound to the chromatographic core in such a way as to blend with thesurface of the chromatographic core resulting in a gradation ofmaterials and no discrete internal core surface. In certain embodiments,the chromatographic surface material may be the same or different fromthe material of the chromatographic core and may exhibit differentphysical or physiochemical properties from the chromatographic core,including, but not limited to, pore volume, surface area, average porediameter, carbon content or hydrolytic pH stability.

The composition of the chromatographic surface material and thechromatographic core material (if present) may be varied by one ofordinary skill in the art to provide enhanced chromatographicselectivity, enhanced column chemical stability, enhanced columnefficiency, and/or enhanced mechanical strength. Similarly, thecomposition of the surrounding material provides a change inhydrophilic/lipophilic balance (HLB), surface charge (e.g., isoelectricpoint or silanol pKa), and/or surface functionality for enhancedchromatographic separation. Furthermore, in some embodiments, thecomposition of the chromatographic material may also provide a surfacefunctionality for available for further surface modification.

The ionizable modifiers and the hydrophobic surface groups of the HPCMsof the invention can be prepared using known methods. Some of theionizable modifier reagents are commercially available. For examplesilanes having amino alkyl trialkoxysilanes, methyl amino alkyltrialkoxysilanes, and pyridyl alkyl trialkoxysilanes are commerciallyavailable. Other silanes such as chloropropyl alkyl trichlorosilane andchloropropyl alkyl trialkoxysilane are also commercially available.These can be bonded and reacted with imidazole to create imidazolylalkyl silyl surface species, or bonded and reacted with pyridine tocreate pyridyl alkyl silyl surface species. Other acidic modifiers arealso commercially available, including, but not limited to,sulfopropyltrisilanol, carboxyethylsilanetriol,2-(carbomethoxy)ethylmethyldichlorosilane,2-(carbomethoxy)ethyltrichlorosilane,2-(carbomethoxy)ethyltrimethoxysilane,n-(trimethoxysilylpropyl)ethylenediamine, triacetic acid,(2-diethylphosphatoethyl)triethoxysilane,2-(chlorosulfonylphenyl)ethyltrichlorosilane, and2-(chlorosulfonylphenyl)ethyltrimethoxysilane.

It is known to one skilled in the art to synthesize these types ofsilanes using common synthetic protocols, including grinard reactionsand hydrosilylations. Products can be purified by chromatography,recrystallization or distillation.

Other additives such as isocyanates are also commercially available orcan be synthesized by one skilled in the art. A common isocyanateforming protocol is the reaction of a primary amine with phosgene or areagent known as Triphosgene.

The ionizable modifier can contain a carboxylic acid group, a sulfonicacid group, a phosphoric acid group, a boronic acid group, an aminogroup, an imido group, an amido group, a pyridyl group, an imidazolylgroup, an ureido group, a thionyl-ureido group or an aminosilane group.

In other aspects the ionizable modifier reagent may be selected fromgroups formula (I)

the formula (II)

the formula (III)

wherein

m is an integer from 1-8;

v is 0 or 1;

when v is 0, m′ is 0;

when v is 1, m′ is an integer from 1-8;

Z represents a chemically reactive group, including (but not limited to)

—OH, —OR⁶, amine, alkylamine, dialkylamine, isocyanate, acyl chloride,triflate, isocyanate, thiocyanate, imidazole carbonate, NHS-ester,carboxylic acid, ester, epoxide, alkyne, alkene, azide, —Br, —Cl or —I;

Y is an embedded polar functionality;

each occurrence of R¹ independently represents a chemically reactivegroup on silicon, including (but not limited to) —H, —OH, —OR⁶,dialkylamine, triflate, Br, Cl, I, vinyl, alkene, or —(CH₂)_(m″)Q;

each occurrence of Q is —OH, —OR⁶, amine, alkylamine, dialkylamine,isocyanate, acyl chloride, triflate, isocyanate, thiocyanate, imidazolecarbonate, NHS-ester, carboxylic acid, ester, epoxide, alkyne, alkene,azide, —Br, —Cl, or —I;

m″ is an integer from 1-8;

p is an integer from 1-3;

each occurrence of R¹′ independently represents F, C₁-C₁₈ alkyl, C₂-C₁₈alkenyl, C₂-C₁₈ alkynyl, C₃-C₁₈ cycloalkyl, C₁-C₁₈ heterocycloalkyl,C₅-C₁₈ aryl, C₅-C₁₈ aryloxy, or C₁-C₁₈ heteroaryl, fluoroalkyl, orfluoroaryl;

each occurrence of R², R^(2′), R³ and R^(3′) independently representshydrogen, C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, C₂-C₁₈ alkynyl, C₃-C₁₈cycloalkyl, C₂-C₁₈ heterocycloalkyl, C₅-C₁₈ aryl, C₅-C₁₈ aryloxy, orC₁-C₁₈ heteroaryl, —Z, or a group having the formula —Si(R′)_(b)R″_(a)or —C(R′)_(b)R″_(a);

a and b each represents an integer from 0 to 3 provided that a+b=3;

R′ represents a C₁-C₆ straight, cyclic or branched alkyl group;

R″ is a functionalizing group selected from the group consisting ofalkyl, alkenyl, alkynyl, aryl, cyano, amino, diol, nitro, ester, acation or anion exchange group, an alkyl or aryl group containing anembedded polar functionality and a chiral moiety.

R⁴ represents hydrogen, C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, C₂-C₁₈ alkynyl,C₃-C₁₈ cycloalkyl, C₁-C₁₈ heterocycloalkyl, C₅-C₁₈ aryl, C₅-C₁₈ aryloxy,or C₁-C₁₈ heteroaryl;

R⁵ represents hydrogen, C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, C₂-C₁₈ alkynyl,C₃-C₁₈ cycloalkyl, C₁-C₁₈ heterocycloalkyl, C₅-C₁₈ aryl, C₅-C₁₈ aryloxy,or C₁-C₁₈ heteroaryl;

each occurrence of R⁶ independently represents C₁-C₁₈ alkyl, C₂-C₁₈alkenyl, C₂-C₁₈ alkynyl, C₃-C₁₈ cycloalkyl, C₁-C₁₈ heterocycloalkyl,C₅-C₁₈ aryl, C₅-C₁₈ aryloxy, or C₁-C₁₈ heteroaryl;

Het represents a heterocyclic or heteroaryl ring system comprising atleast one nitrogen atom; and

A represents an acidic ionizable modifier moiety or a dual chargeionizable modifier moiety.

In certain aspects, where the ionizable modifying reagent is selectedfrom formulas (I), (II) or (III),

m is 2 or 3.

In some aspects, where the ionizable modifying reagent is selected fromformulas (I), (II) or (III), R¹ represents Cl, —OH, dialkylamino,methoxy or ethoxy.

In certain aspects, where the ionizable modifying reagent is selectedfrom formulas (I), (II) or (III), R^(1′) represents, methyl, ethyl,isobutyl, isopropyl or tert-butyl.

In other aspects where the ionizable modifying reagent is selected fromformulas (I), (II) or (III), each occurrence of R² and R³ representshydrogen.

In other aspects where the ionizable modifying reagent is selected fromformulas (I), (II) or (III), each occurrence of R^(2′) and R^(3′)represents hydrogen.

In other aspects where the ionizable modifying reagent is selected fromformula each of R⁴ and R⁵ represents hydrogen.

In still other aspects where the ionizable modifying reagent is selectedfrom formulas (II), Het is pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl,piperidinyl, piperizinyl, hexahydropyrimidinyl, pyrrolyl, pyrazolyl,imidazolyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl or triazinyl.

In other aspects where the ionizable modifying reagent is selected fromformulas (I), (II) or V is 1, m′ is 3, and each occurrence of R²,R^(2′), R³ and R^(3′) is hydrogen. In certain aspects, where theionizable modifying reagent is selected from formulas (I), (II) or(III), V is 1, m′ is 3, and each occurrence of R², R^(2′), R³ and R^(3′)is hydrogen, Y is carbamate, carbonate, amide, urea, ether, thioether,sulfinyl, sulfoxide, sulfonyl, thiourea, thiocarbonate, thiocarbamate ortriazole.

In yet other embodiments, the ionizable modifier isaminopropyltriethoxysilane, aminopropyltrimethoxysilane,2-(2-(trichlorosilyl)ethyl)pyridine, 2-(2-(trimethoxy)ethyl)pyridine,2-(2-(triethoxy)ethyl)pyridine, 2-(4-pyridylethyl)triethoxysilane,2-(4-pyridylethyl)trimethoxysilane, 2-(4-pyridylethyl)trichlorosilane,chloropropyltrimethoxysilane, chloropropyltrichlorosilane,chloropropyltrichlorosilane, chloropropyltriethoxysilane,imidazolylpropyltrimethoxysilane, imidazolylpropyltriethoxysilane,imidazolylpropyl trichlorosilane, sulfopropyltrisilanol,carboxyethylsilanetriol, 2-(carbomethoxy)ethylmethyldichlorosilane,2-(carbomethoxy)ethyltrichlorosilane,2-(carbomethoxy)ethyltrimethoxysilane,n-(trimethoxysilylpropyl)ethylenediamine triacetic acid,(2-diethylphosphatoethyl)triethoxysilane,3-mercaptopropyltriethoxysilane, 3-mercaptopropyltrimethoxysilane,bis[3-(triethoxysilyl)propyl]disulfide,bis[3-(triethoxysilyl)propyl]tetrasulfide,2,2-dimethoxy-1-thia-2-silacyclopentane,bis(trichlorosilylethyl)phenylsulfonyl chloride,2-(chlorosulfonylphenyl)ethyltrichlorosilane,2-(chlorosulfonylphenyl)ethyltrimethoxysilane,2-(ethoxysulfonylphenyl)ethyltrimethoxysilane,2-(ethoxysulfonylphenyl)ethyltrimethoxysilane,2-(ethoxysulfonylphenyl)ethyltrichlorosilane, sulphonic acidphenethyltrisilanol, (triethoxysilyl ethyl)phenyl phosphonic aciddiethyl ester, (trimethoxysilyl ethyl)phenyl phosphonic acid diethylester, (trichlorosilyl ethyl)phenyl phosphonic acid diethyl ester,phosphonic acid phenethyltrisilanol, N-(3-trimethoxysilylpropyl)pyrrole,N-(3-triethoxysilylpropyl)-4,5-dihydroimidazole,bis(methyldimethoxysilylpropyl)-N-methylamine,tris(triethoxysilylpropyl)amine,bis(3-trimethoxysilylpropyl)-N-methylamine,(N,N-diethyl-3-aminopropyl)trimethoxysilane,N-(hydroxyethyl)-N-methylaminopropyltrimethoxysilane,3-(N,N-dimethylaminopropyl)trimethoxysilane,bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane,N,N′-bis(hydroxyethyl)-N,N′-bis(trimethoxysilylpropyl)ethylenediamine,or N,N-dimethyl-3-aminopropylmethyldimethoxysilane.

In certain embodiments, when the ionizable modifier is of the formula(II), the acidic ionizable modifiers is a protected or deprotected formsof trisilanol, trialkoxysilane or trichlorosilane; or a salt of sulfonicacid alkyl silanes, sulfonic acid phenylalkyl silanes, sulfonic acidbenzylalkyl silanes, sulfonic acid phenyl silanes, sulfonic acid benzylsilanes, carboxylic acid alkyl silanes, carboxylic acid phenylalkylsilanes, carboxylic acid benzylalkyl silanes, carboxylic acid phenylsilanes, carboxylic acid benzyl silanes, phosphoric acid alkyl silanes,phosphonic acid phenylalkyl silanes, phosphonic acid benzylalkylsilanes, phosphonic acid phenyl silanes, phosphonic acid benzyl silanes,boronic acid alkyl silanes, boronic acid phenylalkyl silanes, boronicacid benzylalkyl silanes, boronic acid phenyl silanes, boronic acidbenzyl silanes.

In certain embodiments, when the ionizable modifier is of the formula(III), the acidic ionizable modifiers is a protected or deprotectedversion or a salt of sulfonic acid alkyl isocyanates, sulfonic acidphenylalkyl isocyanates, sulfonic acid benzylalkyl isocyanates, sulfonicacid phenyl isocyanates, sulfonic acid benzyl isocyanates carboxylicacid alkyl isocyanates, carboxylic acid phenylalkyl isocyanates,carboxylic acid benzylalkyl isocyanates, carboxylic acid phenylisocyanates, carboxylic acid benzyl isocyanates, phosphoric acid alkylisocyanates, phosphonic acid phenylalkyl isocyanates, phosphonic acidbenzylalkyl isocyanates, phosphonic acid phenyl isocyanates, phosphonicacid benzyl isocyanates, boronic acid alkyl isocyanates, boronic acidphenylalkyl isocyanates, boronic acid benzylalkyl isocyanates, boronicacid phenyl isocyanates, or boronic acid benzyl isocyanates.

In certain embodiments, when the ionizable modifier reagent is selectedfrom formula (III), A represents a dual charge ionizable modifiermoiety. While not limited to theory; the dual charge ionizable modifiermoiety has two sub-groups that can display opposite charges. Under someconditions the dual charge ionizable modifier moiety can act similarlyto a zwitterions and ampholytes to display both a positive and negativecharge and maintain a zero net charge. Under other conditions the dualcharge ionizable modifier moiety may only have one group ionized and maydisplay a net positive or negative charge. Dual charge ionizablemodifier moieties include, but are not limited to, alkyl, branchedalkyl, aryl, cyclic, polyaromatic, polycyclic, hetrocyclic andpolyheterocyclic groups that can display a positive charge (commonly ona nitrogen or oxygen atom), and a negative charge through an acidicgroup that includes a carboxylic, sulfonic, phosphonic or boronic acid.Alternatively, some metal containing complexes can display both positiveand negative charges. Dual charge ionizable modifier moieties may alsoinclude, but are not limited to zwitterions, ampholyte, amino acid,aminoalkyl sulfonic acid, aminoalkyl carboxylic acid, mono anddi-methylaminoalkyl sulfonic acid, mono and di-methylaminoalkylcarboxylic acid, pyridinium alkyl sulfonic acid, and pyridinium alkylcarboxylic acid groups. Alternatively the dual charge ionizable modifiermoiety may be 2-(N-morpholino)ethanesulfonic acid,3-(N-morpholino)propanesulfonic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), piperazine-N,N′-bis(2-ethanesulfonic acid),N-cyclohexyl-3-aminopropanesulfonic acid,N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid,3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate,6-Methyl-9,10-didehydro-ergoline-8-carboxylic acid,phenolsulfonphthalein, betaines, quinonoids,N,N-bis(2-hydroxyethyl)glycine, and N-[tris(hydroxymethyl)methyl]glycinegroups.

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl substituted cycloalkyl groups and cycloalkylsubstituted alkyl groups. In certain embodiments, a straight chain orbranched chain alkyl has 30 or fewer carbon atoms in its backbone, e.g.,C1-C30 for straight chain or C3-C30 for branched chain. In certainembodiments, a straight chain or branched chain alkyl has 20 or fewercarbon atoms in its backbone, e.g., C1-C20 for straight chain or C3-C20for branched chain, and more preferably 18 or fewer. Likewise, preferredcycloalkyls have from 4-10 carbon atoms in their ring structure and morepreferably have 4-7 carbon atoms in the ring structure. The term “loweralkyl” refers to alkyl groups having from 1 to 6 carbons in the chainand to cycloalkyls having from 3 to 6 carbons in the ring structure.

Moreover, the term “alkyl” (including “lower alkyl”) as used throughoutthe specification and claims includes both “unsubstituted alkyls” and“substituted alkyls”, the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example,halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,phosphinato, cyano, amino (including alkyl amino, dialkylamino,arylamino, diarylamino and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. It willbe understood by those skilled in the art that the moieties substitutedon the hydrocarbon chain can themselves be substituted, if appropriate.Cycloalkyls can be further substituted, e.g., with the substituentsdescribed above. An “aralkyl” moiety is an alkyl substituted with anaryl, e.g., having 1 to 3 separate or fused rings and from 6 to about 18carbon ring atoms, e.g., phenylmethyl(benzyl).

The term “amino,” as used herein, refers to an unsubstituted orsubstituted moiety of the formula —NRaRb, in which Ra and Rb are eachindependently hydrogen, alkyl, aryl, or heterocyclyl, or Ra and Rb,taken together with the nitrogen atom to which they are attached, form acyclic moiety having from 3 to 8 atoms in the ring. Thus, the term“amino” includes cyclic amino moieties such as piperidinyl orpyrrolidinyl groups, unless otherwise stated. An “amino-substitutedamino group” refers to an amino group in which at least one of Ra andRb, is further substituted with an amino group.

The term “aromatic group” includes unsaturated cyclic hydrocarbonscontaining one or more rings. Aromatic groups include 5- and 6-memberedsingle-ring groups which may include from zero to four heteroatoms, forexample, benzene, pyrrole, furan, thiophene, imidazole, oxazole,thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine andpyrimidine and the like. The aromatic ring may be substituted at one ormore ring positions with, for example, a halogen, a lower alkyl, a loweralkenyl, a lower alkoxy, a lower alkylthio, a lower alkylamino, a loweralkylcarboxyl, a nitro, a hydroxyl, —CF₃, —CN, or the like.

The term “aryl” includes 5- and 6-membered single-ring aromatic groupsthat may include from zero to four heteroatoms, for example,unsubstituted or substituted benzene, pyrrole, furan, thiophene,imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine,pyridazine and pyrimidine and the like. Aryl groups also includepolycyclic fused aromatic groups such as naphthyl, quinolyl, indolyl andthe like. The aromatic ring can be substituted at one or more ringpositions with such substituents, e.g., as described above for alkylgroups. Suitable aryl groups include unsubstituted and substitutedphenyl groups. The term “aryloxy” as used herein means an aryl group, asdefined above, having an oxygen atom attached thereto. The term“aralkoxy” as used herein means an aralkyl group, as defined above,having an oxygen atom attached thereto. Suitable aralkoxy groups have 1to 3 separate or fused rings and from 6 to about 18 carbon ring atoms,e.g., O-benzyl.

The term “chiral moiety” is intended to include any functionality thatallows for chiral or stereoselective syntheses. Chiral moieties include,but are not limited to, substituent groups having at least one chiralcenter, natural and unnatural amino-acids, peptides and proteins,derivatized cellulose, macrocyclic antibiotics, cyclodextrins, crownethers, and metal complexes.

The term “embedded polar functionality” is a functionality that providesan integral polar moiety such that the interaction with basic samplesdue to shielding of the unreacted silanol groups on the silica surfaceis reduced. Embedded polar functionalities include, but are not limitedto carbonate, amide, urea, ether, thioether, sulfinyl, sulfoxide,sulfonyl, thiourea, thiocarbonate, thiocarbamate, ethylene glycol,heterocyclic, triazole functionalities or carbamate functionalities suchas disclosed in U.S. Pat. No. 5,374,755, and chiral moieties.

The term “heterocyclic group” includes closed ring structures in whichone or more of the atoms in the ring is an element other than carbon,for example, nitrogen, sulfur, or oxygen. Heterocyclic groups can besaturated or unsaturated and heterocyclic groups such as pyrrole andfuran can have aromatic character. They include fused ring structuressuch as quinoline and isoquinoline. Other examples of heterocyclicgroups include pyridine and purine. Heterocyclic groups can also besubstituted at one or more constituent atoms with, for example, ahalogen, a lower alkyl, a lower alkenyl, a lower alkoxy, a loweralkylthio, a lower alkylamino, a lower alkylcarboxyl, a nitro, ahydroxyl, —CF₃, —CN, or the like. Suitable heteroaromatic andheteroalicyclic groups generally will have 1 to 3 separate or fusedrings with 3 to about 8 members per ring and one or more N, O or Satoms, e.g. coumarinyl, quinolinyl, pyridyl, pyrazinyl, pyrimidyl,furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl,benzofuranyl, benzothiazolyl, tetrahydrofuranyl, tetrahydropyranyl,piperidinyl, morpholino and pyrrolidinyl.

In some aspects, the ratio of the hydrophobic surface group:ionizablemodifier in the HPCM of the invention is from about 2.5:1 to about350:1; from about 3:1 to about 200:1; from about 4:1 to about 150:1;from about 4:1 to about 35:1; from about 5:1 to about 25:1; from about5:1 to about 22:1; from about 20:1 to about 100:1; or from about 25:1 toabout 100:1.

In some embodiments, the ratio of the hydrophobic surfacegroup:ionizable modifier in the HPCM of the invention is from about 4:1to about 150:1; from about 20:1 to about 100:1; or from about 25:1 toabout 100:1.

In other aspects, the concentration of ionizable modifier in the HPCM ofthe invention is less than about 0.5 μmol/m²; less than about 0.4μmol/m²; less than about 0.3 μmol/m²; from about 0.01 μmol/m² to about0.5 μmol/m²; from about 0.01 μmol/m² to about 0.4 μmol/m²; or from about0.03 μmol/m² to about 0.3 μmol/m².

In other embodiments, the concentration of ionizable modifier in theHPCM of the invention is less than about 0.5 μmol/m²; less than about0.4 μmol/m²; less than about 0.3 mmol/m²; from about 0.01 μmol/m² toabout 0.5 μmol/m²; from about 0.1 μmol/m² to about 0.4 μmol/m²; or fromabout 0.2 μmol/m² to about 0.3 μmol/m².

In another aspect, the hydrophobic surface group of the HPCM of theinvention is a C₄ to C₃₀ bonded phase. In certain aspects, thehydrophobic surface group is a C₁ bonded phase. In other aspects, thehydrophobic surface group is an aromatic, phenylalkyl, fluoro-aromatic,phenylhexyl, pentafluorophenylalkyl or chiral bonded phase. In stillother aspects, the hydrophobic surface group is an embedded polar bondedphase.

In certain aspects, the HPCM of the invention may be in the form of aparticle, a granular material, a monolith, a superficially porousmaterial, a superficially porous particle, a superficially porousmonolith, or a superficially porous layer for open tubularchromatography.

In certain aspects, the HPCM of the invention may be in inorganicmaterial (e.g., silica, alumina, titania, zirconia), a hybridorganic/inorganic material, an inorganic material (e.g., silica,alumina, titania, zirconia) with a hybrid surface layer, a hybridmaterial with an inorganic (e.g., silica, alumina, titania, zirconia)surface layer, or a hybrid material with a different hybrid surfacelayer. In other aspects, the HPCM of the invention may have ordered porestructure, non-periodic pore structuring, non-crystalline or amorphouspore structuring or substantially disordered pore structuring.

The term “substantially disordered” refers to a lack of pore orderingbased on x-ray powder diffraction analysis. Specifically, “substantiallydisordered” is defined by the lack of a peak at a diffraction angle thatcorresponds to a d value (or d-spacing) of at least 1 nm in an x-raydiffraction pattern.

In still another aspect, the HPCM of the invention has a quantifiedsurface coverage ratio, B/A, from about 2.5 to about 300 wherein Arepresents the ionizable modifier and B represents the hydrophobicgroup. In certain aspects, the quantified surface coverage ratio, B/A,is from about 3 to about 200, from about 4 to about 35 or from about 5to about 22.

In another aspect, the hydrophobic surface group of the HPCM of theinvention is a C4 to C18 bonded phase. In certain aspects, thehydrophobic surface group is a C18 bonded phase. In still other aspects,the hydrophobic surface group is an embedded polar bonded phase. Inother aspects, the hydrophobic surface group is an aromatic,phenylalkyl, fluoro-aromatic, phenylhexyl, or pentafluorophenylalkylbonded phase. In another aspect, the hydrophobic surface group is aC₄-C₃₀, embedded polar, chiral, phenylalkyl, or pentafluorophenylbonding or coating.

In certain embodiments, the HPCM of the invention may be in the form ofa particle, a monolith or a superficially porous material. In certainother aspects, the HPCM of the invention is a non-porous material.

In certain aspects, the HPCM of the invention may be an inorganicmaterial (e.g., silica), a hybrid organic/inorganic material, aninorganic material (e.g., silica) with a hybrid surface layer, a hybridparticle with a inorganic (e.g., silica) surface layer, or a hybridparticle with a different hybrid surface layer.

In one embodiment, the HPCM of the invention does not havechromatographically enhancing pore geometry. In another embodiment, theHPCM of the invention has chromatographically enhancing pore geometry.

The language “chromatographically-enhancing pore geometry” includes thegeometry of the pore configuration of the presently-disclosed materials,which has been found to enhance the chromatographic separation abilityof the material, e.g., as distinguished from other chromatographic mediain the art. For example, a geometry can be formed, selected orconstructed, and various properties and/or factors can be used todetermine whether the chromatographic separations ability of thematerial has been “enhanced”, e.g., as compared to a geometry known orconventionally used in the art. Examples of these factors include highseparation efficiency, longer column life and high mass transferproperties (as evidenced by, e.g., reduced band spreading and good peakshape.) These properties can be measured or observed usingart-recognized techniques. For example, thechromatographically-enhancing pore geometry of the present porousinorganic/organic hybrid materials is distinguished from the prior artmaterials by the absence of “ink bottle” or “shell shaped” pore geometryor morphology, both of which are undesirable because they, e.g., reducemass transfer rates, leading to lower efficiencies.

Chromatographically-enhancing pore geometry is found in hybrid materialscontaining only a small population of micropores. A small population ofmicropores is achieved in hybrid materials when all pores of a diameterof about <34 Å contribute less than about 110 m²/g to the specificsurface area of the material. Hybrid materials with such a low microporesurface area (MSA) give chromatographic enhancements including highseparation efficiency and good mass transfer properties (as evidencedby, e.g., reduced band spreading and good peak shape). Micropore surfacearea (MSA) is defined as the surface area in pores with diameters lessthan or equal to 34 Å, determined by multipoint nitrogen sorptionanalysis from the adsorption leg of the isotherm using the BJH method.As used herein, the acronyms “MSA” and “MPA” are used interchangeably todenote “micropore surface area”.

The term “functionalizing group” includes organic functional groupswhich impart a certain chromatographic functionality to achromatographic stationary phase.

In certain embodiments, the HPCM of the invention has a surface area ofabout 25 to 1100 m²/g; about 80 to 500 m²/g; or about 120 to 330 m²/g.

In other embodiments, the HPCM of the invention a pore volume of about0.15 to 1.7 cm³/g; or about 0.5 to 1.3 cm³/g.

In certain other embodiments, the HPCM of the invention is non-porous.

In yet other embodiments, the HPCM of the invention has a microporesurface area of less than about 110 m²/g; less than about 105 m²/g; lessthan about 80 m²/g; or less than about 50 m²/g.

In still yet other embodiments, the HPCM of the invention has an averagepore diameter of about 20 to 1500 Å; about 50 to 1000 Å; about 100 to750 Å; or about 150 to 500 Å.

In still yet other aspects, when the HPCM of the invention is in theform of a particle, the HPCM of the invention has an average particlesize of about 0.3-100 μm; about 0.5-20 μm; 0.8-10 μm; or about 1.0-3.5μm.

In another embodiment, the HPCM of the invention is hydrolyticallystable at a pH of about 1 to about 14; at a pH of about 10 to about 14;or at a pH of about 1 to about 5.

In another aspect, the invention provides materials as described hereinwherein the HPCM material further comprises a nanoparticle or a mixtureof more than one nanoparticles dispersed within the chromatographicsurface.

The term “nanoparticle” is a microscopic particle/grain or microscopicmember of a powder/nanopowder with at least one dimension less thanabout 100 nm, e.g., a diameter or particle thickness of less than about100 nm (0.1 mm), which may be crystalline or noncrystalline.Nanoparticles have properties different from, and often superior tothose of conventional bulk materials including, for example, greaterstrength, hardness, ductility, sinterability, and greater reactivityamong others. Considerable scientific study continues to be devoted todetermining the properties of nanomaterials, small amounts of which havebeen synthesized (mainly as nano-size powders) by a number of processesincluding colloidal precipitation, mechanical grinding, and gas-phasenucleation and growth. Extensive reviews have documented recentdevelopments in nano-phase materials, and are incorporated herein byreference thereto: Gleiter, H. (1989) “Nano-crystalline materials,”Prog. Mater. Sci. 33:223-315 and Siegel, R. W. (1993) “Synthesis andproperties of nano-phase materials,” Mater. Sci. Eng. A168:189-197. Incertain embodiments, the nanoparticles comprise oxides or nitrides ofthe following: silicon carbide, aluminum, diamond, cerium, carbon black,carbon nanotubes, zirconium, barium, cerium, cobalt, copper, europium,gadolinium, iron, nickel, samarium, silicon, silver, titanium, zinc,boron, and mixtures thereof. In certain embodiments, the nanoparticlesof the present invention are selected from diamonds, zirconium oxide(amorphous, monoclinic, tetragonal and cubic forms), titanium oxide(amorphous, anatase, brookite and rutile forms), aluminum (amorphous,alpha, and gamma forms), and boronitride (cubic form). In particularembodiments, the nanoparticles of the present invention are selectedfrom nano-diamonds, silicon carbide, titanium dioxide (anatase form),cubic-boronitride, and any combination thereof. Moreover, in particularembodiments, the nanoparticles may be crystalline or amorphous. Inparticular embodiments, the nanoparticles are less than or equal to 100mm in diameter, e.g., less than or equal to 50 mm in diameter, e.g.,less than or equal to 20 mm in diameter.

Moreover, it should be understood that the nanoparticles that arecharacterized as dispersed within the composites of the invention areintended to describe exogenously added nanoparticles. This is incontrast to nanoparticles, or formations containing significantsimilarity with putative nanoparticles, that are capable of formation insitu, wherein, for example, macromolecular structures, such asparticles, may comprise an aggregation of these endogenously created.

In certain embodiments, the nanoparticle is present in <20% by weight ofthe nanocomposite, <10% by weight of the nanocomposite, or <5% by weightof the nanocomposite.

In other embodiments, the nanoparticle is crystalline or amorphous andmay be silicon carbide, aluminum, diamond, cerium, carbon black, carbonnanotubes, zirconium, barium, cerium, cobalt, copper, europium,gadolinium, iron, nickel, samarium, silicon, silver, titanium, zinc,boron, oxides thereof, or a nitride thereof. In particular embodiments,the nanoparticle is a substance which comprises one or more moietiesselected from the group consisting of nano-diamonds, silicon carbide,titanium dioxide, and cubic-boronitride. In other embodiments, thenanoparticles may be less than or equal to 200 nm in diameter, less thanor equal to 100 nm in diameter, less than or equal to 50 nm in diameter,or less than or equal to 20 nm in diameter.

The HPCM materials of the invention may further be surface modified.

“Surface modifiers” include (typically) organic functional groups whichimpart a certain chromatographic functionality to a chromatographicstationary phase. The porous inorganic/organic hybrid materials possessboth organic groups and silanol groups which may additionally besubstituted or derivatized with a surface modifier.

The language “surface modified” is used herein to describe the compositematerial of the present invention that possess both organic groups andsilanol groups which may additionally be substituted or derivatized witha surface modifier. “Surface modifiers” include (typically) organicfunctional groups which impart a certain chromatographic functionalityto a chromatographic stationary phase. Surface modifiers such asdisclosed herein are attached to the base material, e.g., viaderivatization or coating and later crosslinking, imparting the chemicalcharacter of the surface modifier to the base material. In oneembodiment, the organic groups of a hybrid material, react to form anorganic covalent bond with a surface modifier. The modifiers can form anorganic covalent bond to the material's organic group via a number ofmechanisms well known in organic and polymer chemistry including but notlimited to nucleophilic, electrophilic, cycloaddition, free-radical,carbene, nitrene, and carbocation reactions. Organic covalent bonds aredefined to involve the formation of a covalent bond between the commonelements of organic chemistry including but not limited to hydrogen,boron, carbon, nitrogen, oxygen, silicon, phosphorus, sulfur, and thehalogens. In addition, carbon-silicon and carbon-oxygen-silicon bondsare defined as organic covalent bonds, whereas silicon-oxygen-siliconbonds that are not defined as organic covalent bonds. A variety ofsynthetic transformations are well known in the literature, see, e.g.,March, J. Advanced Organic Chemistry, 3rd Edition, Wiley, New York,1985.

Thus, in one embodiment, the material as described herein may be surfacemodified with a surface modifier having the formula Z_(a)(R′)_(b)Si—R″,where Z=Cl, Br, I, C₁-C₅ alkoxy, dialkylamino ortrifluoromethanesulfonate; a and b are each an integer from 0 to 3provided that a+b=3; R′ is a C₁-C₆ straight, cyclic or branched alkylgroup, and R″ is a functionalizing group.

In another embodiment, the materials have been surface modified bycoating with a polymer.

In certain embodiments, R′ is selected from the group consisting ofmethyl, ethyl, propyl, isopropyl, butyl, t-butyl, sec-butyl, pentyl,isopentyl, hexyl and cyclohexyl. In other embodiments, R′ is selectedfrom the group consisting of alkyl, alkenyl, alkynyl, aryl, cyano,amino, diol, nitro, ester, a cation or anion exchange group, an alkyl oraryl group containing an embedded polar functionality and a chiralmoiety. In certain embodiments, R′ is selected from the group consistingof aromatic, phenylalkyl, fluoroaromatic, phenylhexyl,pentafluorophenylalkyl and chiral moieties.

In one embodiment, R″ is a C₁-C₃₀ alkyl group. In a further embodiment,R″ comprises a chiral moiety. In another further embodiment, R″ is aC₁-C₂₀ alkyl group.

In certain embodiments, the surface modifier comprises an embedded polarfunctionality. In certain embodiments, such embedded polar functionalityincludes carbonate, amide, urea, ether, thioether, sulfinyl, sulfoxide,sulfonyl, thiourea, thiocarbonate, thiocarbamate, ethylene glycol,heterocyclic, or triazole functionalities. In other embodiments, suchembedded polar functionality includes carbamate functionalities such asdisclosed in U.S. Pat. No. 5,374,755, and chiral moieties. Such groupsinclude those of the general formula

wherein l, m, o, r and s are 0 or 1, n is 0, 1, 2 or 3 p is 0, 1, 2, 3or 4 and q is an integer from 0 to 19; R³ is selected from the groupconsisting of hydrogen, alkyl, cyano and phenyl; and Z, R′, a and b aredefined as above. Preferably, the carbamate functionality has thegeneral structure indicated below:

wherein R⁵ may be, e.g., cyanoalkyl, t-butyl, butyl, octyl, dodecyl,tetradecyl, octadecyl, or benzyl. Advantageously, R⁵ is octyl, dodecyl,or octadecyl.

In certain embodiments, the surface modifier is selected from the groupconsisting of phenylhexyltrichlorosilane,pentafluorophenylpropyltrichlorosilane, octyltrichlorosilane,octadecyltrichlorosilane, octyldimethylchlorosilane andoctadecyldimethylchlorosilane. In some embodiments, the surface modifieris selected from the group consisting of octyltrichlorosilane andoctadecyltrichlorosilane. In other embodiments, the surface modifier isselected from the group consisting of an isocyanate or1,1′-carbonyldiimidazole (particularly when the hybrid group contains a(CH₂)₃OH group).

In another embodiment, the material has been surface modified by acombination of organic group and silanol group modification.

In still another embodiment, the material has been surface modified by acombination of organic group modification and coating with a polymer. Ina further embodiment, the organic group comprises a chiral moiety.

In yet another embodiment, the material has been surface modified by acombination of silanol group modification and coating with a polymer.

In other embodiments, the material has been surface modified viaformation of an organic covalent bond between the particle's organicgroup and the modifying reagent.

In still other embodiments, the material has been surface modified by acombination of organic group modification, silanol group modificationand coating with a polymer.

In another embodiment, the material has been surface modified by silanolgroup modification.

In certain embodiments, the surface modified layer may be porous ornon-porous.

Another aspect provides a variety of separations devices having astationary phase comprising the HPCM materials as described herein. Theseparations devices include, e.g., chromatographic columns, thin layerplates, filtration membranes, sample cleanup devices and microtiterplates.

The HPCM Materials impart to these devices improved lifetimes because oftheir improved stability. Thus, in a particular aspect, the inventionprovides a chromatographic column having improved lifetime, comprising

a) a column having a cylindrical interior for accepting a packingmaterial, and

b) a packed chromatographic bed comprising the high puritychromatographic material as described herein.

In another particular aspect, the invention provides a chromatographicdevice, comprising

a) an interior channel for accepting a packing material and

b) a packed chromatographic bed comprising the high puritychromatographic material as described herein.

The invention also provides for a kit comprising the HPCM materials asdescribed herein, as described herein, and instructions for use. In oneembodiment, the instructions are for use with a separations device,e.g., chromatographic columns, thin layer plates, filtration membranes,sample cleanup devices and microtiter plates.

Mass Spectroscopy

Further to the summary above, the technology includes analyzing thecomponent of the first fraction by mass spectroscopy, therebyidentifying the polypeptide, if present, using a signal corresponding toa sequence fragment ion from the polypeptide. The analysis step of thetechnology works in concert with the pre-treatment and separation steps,to provide a high resolution tool for studying polypeptides.

In general, the first fraction should include a sequence that isindicative of, and specific to the polypeptide analyte. For example, thecomponent of the first fraction can include the polypeptide analyte inan undigested, or essentially undigested, state.

Accordingly, MS analysis can produce a sequence fragment ion from thepolypeptide that is indicative of, and specific to the polypeptideanalyte. For example, the signal corresponds to an intact multiplycharged precursor fragment selected in a first quadrupole and itscorresponding sequence fragment ion selected in a final quadrupole. Inthe examples of insulin discussed below, the sequence fragment ionexhibits an m/z >800. Where the method is used on another polypeptide,the sequence fragment ion can exhibit an m/z that is characteristic ofthat polypeptide in an undigested, or essentially undigested state. Inan alternative embodiment, the polypeptide can be digested, as long asthe sequence fragment ion is indicative of, and specific to thepolypeptide analyte.

In some embodiments (e.g., where one might typically rely on a singleprecursor to generate fragments for small molecule analyses), there canbe advantages to selecting multiple precursors to attempt fragmentationof a peptide analyte. Not only can the various charge states fragmentdifferently, but it can be difficult to predict the specificity of aparticular MRM in a sample derived from a biological matrix.Furthermore, there can exist some question as to whether or not therelative abundance of the various charge states changes during analysis,driving the desire to monitor and possibly sum MRMs arising fromdistinct precursors.

FIGS. 22A-22D shows an MS spectrum for four synthetic insulin analogs,Lantus® (insulin glargine), Levemir® (insulin detemir),Novolog/NovoRapid® (insulin aspart), and Apidra® (insulin glulisine).Note that each analog yields a unique selection and pattern ofprecursors, despite their closely related chemical nature. In someembodiments, for each analog, the two or three most abundant precursorswere chosen for fragmentation. CID of the chosen precursors wasperformed over the range of m/z 100-1800. Both the 6+ and 7+ insulinglargine precursors yielded multiple possible fragments, whereas onlythe 6+ charge state of insulin aspart and the 5+ of glulisine anddetemir resulted in fragment ions with sufficient intensity formeaningful quantification. Representative MSMS spectra for each insulinanalog at its optimum collision energy is shown in FIGS. 22A-22D.Fragments chosen for quantification are highlighted with an asterisk.

In some embodiments, under low resolution conditions, there is overlapbetween the isotope patterns of the 5+ and 6+ multiply chargedprecursors of aspart and glulisine (see FIGS. 1, 8A, 13 and 17). Thiscan lead to a lack of specificity if unique fragments are not chosen. Insome embodiments, these same precursors can also overlap with humaninsulin. However, certain fragments arising from human insulin (e.g., atm/z 561, 653, and 226) are also unique (data not shown). At highercollision energies, each of the insulin analogs can produce immonium ionfragments with high intensity. Most yield an intense peak m/z 136, forexample, corresponding to a tyrosine immonium ion. These spectra aresimpler than the MSMS spectra for the insulins at their optimal, lowercollision energies and they are dominated by a single intense fragment,rather than several low intensity fragments. In some embodiments, thechoice of peptide fragment ion is more complicated than simply choosingthe most intense fragment.

In some embodiments, due to the nominal mass resolution limitations ofcertain triple quadrupole instruments, the high concentration of otherpeptides in the sample (possibly closely related), and/or the highchemical background associated with low m/z fragments, specificity inthe endogenous matrix was found to be a critical deciding factor. Forexample, improved sensitivity was observed at higher mass ranges,facilitating the use of fragments such as the m/z 984 for glargine andm/z 1370 for glulisine in quantification. This observed specificitybenefit derived from these larger fragments was central to reducing theoverall demand on the sample preparation, and contributed to the use ofa significantly simpler and faster sample prep scheme than previouslypublished methods.

The mass spectrometer should also be capable of obtaining a signalcorresponding to a sequence fragment ion from the polypeptide. The massspectrometer should also be capable of achieving a desired detectionlimit. For example, the mass spectrometry can employ a triple quadrupolemass spectrometer. The mass spectrometry can be carried out in positiveelectrospray ionisation mode. In certain embodiments, the detectionlimit of the polypeptide of interest, for instance for insulin or aninsulin analog, is 0.25 ng/mL or less. The detection limit of thepolypeptide of interest can be 0.50 ng/mL or less. The detection limitcan be achieved in a specimen of 250 microliters or less. In someembodiments, the detection limit of the polypeptide of interest can be30 pg/mL or less. In some embodiments, the detection limit of thepolypeptide of interest can be 15 pg/mL or less. For instance, in someembodiments, the detection limit of the polypeptide of interest can be30 pg/mL or less, 29 pg/mL or less, 28 pg/mL or less, 27 pg/mL or less,26 pg/mL or less, 25 pg/mL or less, 24 pg/mL or less, 23 pg/mL or less,22 pg/mL or less, 21 pg/mL or less, 20 pg/mL or less, 19 pg/mL or less,18 pg/mL or less, 17 pg/mL or less, 16 pg/mL or less, or 15 pg/mL orless.

The MS can employ RF transmission optics to improve sensitivity, forexample by allowing the user to pull in more ions, filter non-ions, andfocus the ion beam for efficient transmission. In one embodiment, thetechnology employs RF transmission optics by use of an LC/MS/MSapparatus.

Specificity

For a method in accordance with the invention, where several closelyrelated large peptides (e.g., with multiple possible molecular ions) arebeing analyzed in complex matrix using low resolution mass spectrometry,the risk of interference from the MRM of one insulin channel intoanother can be high. Therefore, one must assess the contribution ofanalytes present individually at high concentration, to the response inother channels. In order to evaluate the specificity of the method,samples were fortified individually with one of the four analogs at aconcentration of 500 ng/mL, which is 20× the plasma ULOQ of 25 ng/mL. Inaddition, samples were fortified with human insulin only at 500 ng/mLand 1 μg/mL to assess the impact and potential interference of highlevels of endogenous insulin (as might be present in Type 2 diabetics)on assay specificity.

EXAMPLES

Unless indicated otherwise, all techniques, including the use of kitsand reagents, can be carried out according to the manufacturers'information, methods known in the art, or as described, for example, inTietz Text Book of Clinical Chemistry 3^(rd) Edition (Burtis, C. A. &Ashwood, M. D., Eds.) W. B. Saunders Company, 1999; Guidance forIndustry. Bioanalytical Method Validation. USA: Centre for DrugEvaluation and Research, US Department of Health and Social Services,Food and Drug Administration, 2001; and Sambrook et al., MolecularCloning. A Laboratory Manual, 2^(nd) Edition (1989) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.

In the following four examples, four synthetic insulin analogs, Lantus®(insulin glargine), Levemir® (insulin detemir), Novolog/NovoRapid®(insulin aspart), and Apidra® (insulin glulisine), were analyzed inhuman plasma samples. The fifth example is an example illustrating theuse of this technology with a sample of human plasma obtained from asubject to measure a generic analyte of interest.

Samples were prepared by spiking the synthetic insulin analogs, Lantus®(insulin glargine), Levemir® (insulin detemir), Novolog/NovoRapid®(insulin aspart), and Apidra® (insulin glulisine), in human plasma tofinal concentrations of between 50 pg/mL and 500 ng/mL.

Treatment and extraction of the samples was carried out on a microelution 96-well plate designed for solid phase extraction and having ahydrophilic-lipophilic-balanced reversed-phase sorbent and conditionedwith 200 μL methanol and equilibrated with 200 μL water. Samples werediluted with 250 μL 10 mM TRIS base(2-amino-2-hydroxymethyl-propane-1,3-diol) and then loaded onto theplate. The plates were washed with 200 μL 5% methanol, 1% acetic acid inwater; eluted with 2×25 μL 60% methanol, 10% acetic acid in water; anddiluted with 50 μL water. Recoveries ranged from 82-95%.

Separation was carried out on an ultra high performance liquidchromatography apparatus with a flow-through needle auto sampler usingan ultra high-performance liquid chromatography column (2.1×50 mm, 1.7μm) wherein the chromatographic surface includes a hydrophobic surfacegroup and one or more ionizable modifiers using the followingparameters—Mobile Phase A: 0.1% formic acid in water; Mobile Phase B:0.1% formic acid in acetonitrile; Weak wash: mobile phase A; Strongwash: 50/25/24/1 ACN/IPA/water/formic acid; Flow rate: 0.3 mL/min;Column temperature: 45° C.; Sample Manager temperature: 15° C.;Gradient: 20% B to 65% B in 2 min, to 98% B at 2.1 min, hold for 0.5min, return to initial at 2.7 min; Total cycle time: 3.5 min; Injectionsolvent: methanol and acetic acid; and Injection volume: 10 μL.Representative standard curve statistics for insulin glulisine (A) andinsulin glargine (B) from 50 pg/mL to 500 ng/mL in solvent standards areshown below in Table 1A and 1B:

TABLE 1A Representative standard curve statistics for insulin glulisinefrom 50 pg/mL to 500 ng/mL in solvent standards Expected Calc. NameConc. (ng/mL) Area Conc. (ng/mL) % Dev. 50 pg/mL 0.05 246.6 0.045 −9.5100 pg/mL 0.1 563.7 0.101 1 200 pg/mL 0.2 1090 0.194 −3.2 500 pg/mL 0.52642.8 0.467 −6.7 1 ng/mL 1 5544.9 0.977 −2.3 2 ng/mL 2 11120.5 1.957−2.1 5 ng/mL 5 28094.9 4.942 −1.2 10 ng/mL 10 61467.9 10.811 8.1 20ng/mL 20 117881.6 20.731 3.7 50 ng/mL 50 298668.7 52.523 5 100 ng/mL 100625570.8 110.009 10 500 ng/mL 500 2764219.3 486.092 −2.8

TABLE 1B Representative standard curve statistics tor insulin glarginefrom 50 pg/mL to 500 ng/mL in solvent standards Expected Calc. NameConc. (ng/mL) Area Conc. (ng/mL) % Dev 50 pg/mL 0.05 483.06 0.051 2.4100 pg/mL 0.1 777.31 0.094 −6.4 500 pg/mL 0.5 3748.09 0.522 4.3 1 ng/mL1 6955.55 0.984 −1.6 2 ng/mL 2 15881.66 2.27 13.5 5 ng/mL 5 38498.985.528 10.6 10 ng/mL 10 74175.04 10.668 6.7 20 ng/mL 20 143202.70 20.6123.1 50 ng/mL 50 308271.28 44.393 −11.2 500 ng/mL 500 2734696.00 393.954−21.2

MS analysis was carried out on a triple quadrupole MS using thefollowing parameters—Capillary: 3.00 kV; Desolvation temperature: 600°C.; Source temperature: 150° C. Desolvation flow: 1000 L/hr; and theinsulin analog-specific MRM transitions listed below. MassLynx™ softwarewas used for data acquisition. All peak area integration, regressionanalysis and sample quantification was performed using TargetLynx™software. Peak area ratios (PARs) of the insulin analogs and the bovineinsulin internal standard were determined and calibration curvesgenerated for each of the analogs. Insulin analog concentrations in QCsamples were determined from their PARs against their respectivecalibration lines.

Example 1 Lantus® (Insulin Glargine)

Samples were treated, extracted, separated, and analyzed as describedabove. The results are shown and discussed in connection with FIGS.1-7A-7E below. Analysis of Lantus® (insulin glargine) used the MRMtransitions shown in Table 2 below:

TABLE 2 MRM transitions for Lantus ® (insulin glargine). Cone CollisionMRM Transition Voltage (V) Energy (eV) 867 −> 856 60 18 867 −> 984 60 181011 −> 1164 60 25 1011 −> 1179 60 25

The 867→984 transition was used for quantitation of Lantus® (insulinglargine) based upon MS tuning studies, low detection limit, andlinearity.

FIG. 1 shows a MS scan of Lantus® (insulin glargine) with the 8+, 7+,6+, and 5+ transitions annotated. FIG. 2 shows a MS/MS scan of m/z 867,which is the 7+ precursor of Lantus® (insulin glargine). FIG. 3 shows achromatogram for the 867→984 transition monitoring for Lantus® (insulinglargine) (the A chain plus a y19 B chain ion type) using a 2 ng/mLstandard solution (peak width 3.6 s).

FIG. 4A shows chromatograms for 100 pg/mL (top panel) and FIG. 4B shows50 pg/mL (middle panel) of Lantus® (insulin glargine) and FIG. 4C showssolvent blank (bottom panel). 30% methanol, 10% acetic acid, and 0.05%rat plasma was used as a dilution solvent. FIGS. 4D-4F shows integratedanalyte peaks from the chromatograms shown in FIGS. 4A-4C. The detectionlimit was 50 pg/mL. FIGS. 5A-5C shows the linearity for Lantus® (insulinglargine) in solvent standards from 50 pg/mL to 500 ng/mL.

FIG. 6A shows chromatograms for 10 μL injections of 0.5 ng/mL (toppanel) and 0.2 ng/mL of Lantus® (insulin glargine) extracted from humanplasma and analyzed in accordance with the methods described above. FIG.6C shows a blank of human plasma is also shown for comparison (bottompanel). FIGS. 7A-E shows chromatograms for additional levels andintegration for Lantus® (insulin glargine). Concentrations shown are 5ng/mL (top panel), FIG. 7A, 1 ng/mL (second panel from top), FIG. 7B,0.5 ng/mL (middle panel), FIG. 7C, 0.2 ng/mL (second from bottom), FIG.7D, and blank human plasma (bottom panel) FIG. 7E.

Example 2 Levemir® (Insulin Detemir)

Samples were treated, extracted, separated, and analyzed as describedabove. The results are shown and discussed in connection with FIGS.8A-12A-12C below. Analysis of Levemir® (insulin detemir) used the MRMtransitions shown in Table 3 below:

TABLE 3 MRM transitions for Levemir ® (insulin detemir). Cone CollisionMRM Transition Voltage (V) Energy (eV)  1184 −> 454.4 60 20 1183.8 −>1366.3 60 20

The 1184→454.4 transition was used for quantitation of Levemir® (insulindetemir) (a y2 type ion) based upon MS tuning studies, low detectionlimit, and linearity.

FIG. 8A shows a MS scan of Levemir® (insulin detemir) with the 6+, 5+,and 4+ transitions annotated. FIG. 8B shows a MS/MS scan of m/z 1184,which is the 5+ precursor of Levemir® (insulin detemir).

FIG. 9A shows chromatograms for 200 pg/mL (top panel), 100 pg/mL (secondpanel from top), FIG. 9B, and 50 pg/mL (second panel from bottom), FIG.9C, of Levemir® (insulin detemir) and solvent blank (bottom panel), FIG.9D. 30% methanol, 10% acetic acid, and 0.05% rat plasma was used as adilution solvent. FIGS. 9E-9F shows an integrated analyte peak from thechromatograms shown in FIGS. 9A-9D. The detection limit was 50 pg/mL.FIGS. 10A-10C shows the linearity for Levemir® (insulin detemir) insolvent standards from 50 pg/mL to 500 ng/mL.

FIG. 11A shows chromatograms and peak integrations from 10 μL injectionsof 1 ng/mL (top panel), and 0.5 ng/mL (middle panel), FIG. 11B extractedfrom human plasma and analyzed in accordance with the methods describedabove. A blank human plasma is also shown for comparison (bottom panel),FIG. 11C.

FIG. 12A shows chromatograms and peak integrations from 20 μL injectionsof 0.5 ng/mL (top panel) and 0.2 ng/mL (middle panel), FIG. 12Bextracted from human plasma and analyzed in accordance with the methodsdescribed above. A blank human plasma is also shown for comparison(bottom panel), FIG. 12C.

Example 3 Apidra® (Insulin Glulisine)

Samples were treated, extracted, separated, and analyzed as describedabove. The results are shown and discussed in connection with FIGS.13-16A-16C below. Analysis of Apidra® (insulin glulisine) used the MRMtransitions shown in Table 4 below:

TABLE 4 MRM transitions for Apidra ® (insulin glulisine). Cone CollisionMRM Transition Voltage (V) Energy (eV) 1165 −> 346.2  14 22 1165 −>1161.6 14 22 1165 −> 1369.9 14 22

The 1165→346.2 (a y3 type ion) or 1369.9 transitions were used forquantitation of Apidra® (insulin glulisine) based upon MS tuningstudies, low detection limit, and linearity.

FIG. 13 shows a MS scan of Apidra® (insulin glulisine) with the 6+ and5+ transitions annotated. FIG. 14A shows chromatograms for 100 pg/mL(top panel) and 50 pg/mL (middle panel), FIG. 14B of Apidra® (insulinglulisine) and blank solvent (bottom panel), FIG. 14C. The detectionlimit was 50 pg/mL. FIGS. 15A-15C shows the linearity for Apidra®(insulin glulisine) in solvent standards from 50 pg/mL to 500 ng/mL.

FIG. 16A shows chromatograms and peak integrations of 0.5 ng/mL (toppanel), and 0.2 ng/mL (middle panel), FIG. 16B of Apidra® (insulinglulisine) extracted from human plasma and analyzed in accordance withthe methods described above. A blank human plasma is also shown forcomparison (bottom panel), FIG. 16C.

Example 4 Novolog/Novorapid® (Insulin Aspart)

Samples were treated, extracted, separated, and analyzed as describedabove. The results are shown and discussed in connection with FIGS.17-20A-20C below. Analysis of Novolog/Novorapid® (insulin aspart) usedthe MRM transitions shown in Table 5 below:

TABLE 5 MRM transitions for Novolog/Novorapid ® (insulin aspart). ConeCollision MRM Transition Voltage (V) Energy (eV)  971.6 −> 1139.4 12 18971.8 −> 660.8 60 18 971.6 −> 1146  12 18 1165.7 −> 1395.4 88 28

The 971.8→660.8 transition (a y11 type ion) was used for quantitation ofNovolog/Novorapid® (insulin aspart) based upon MS tuning studies, lowdetection limit, and linearity. Additionally, the 971.6-1139.4transition gives an ion type that is A chain plus b20 B chain.

FIG. 17 shows a MS scan of Novolog/Novorapid® (insulin aspart) with the6+ and 5+ transitions annotated. FIG. 18A shows chromatograms for 200pg/mL (top panel), 100 pg/mL (second panel from top), FIG. 18B, and 50pg/mL (second panel from bottom), FIG. 18C of Novolog/Novorapid®(insulin aspart) and solvent blank (bottom panel), FIG. 18D. FIG. 18Eshows an integrated analyte peak from the chromatograms shown in FIG.18A (top panel) and solvent blank (bottom panel), FIG. 18D. Thedetection limit was 50 pg/mL. FIGS. 19A-19C shows the linearity forNovolog/Novorapid® (insulin aspart) in solvent standards from 50 pg/mLto 500 ng/mL.

FIG. 20A shows chromatograms for injections of 1 ng/mL (top panel) and0.5 ng/mL (middle panel), FIG. 20B of Novolog/Novorapid® (insulinaspart) extracted from human plasma and analyzed in accordance with themethods described above. A blank of human plasma is also shown forcomparison (bottom panel), FIG. 20C.

Example 5 Human Plasma

A 250 μL sample of plasma is obtained from a human subject. Treatmentand extraction of the sample is carried out on a Waters Oasis® HLB microelution 96-well plate designed for solid phase extraction and having ahydrophilic-lipophilic-balanced reversed-phase sorbent and conditionedwith 200 μL methanol equilibrated with 200 μL water. The plasma sampleis diluted with 250 μL 10 mM TRIS base(2-amino-2-hydroxymethyl-propane-1,3-diol) and then loaded onto theplate. The plate is washed with 200 μL 5% methanol, 1% acetic acid inwater and eluted with 2×25 μL 60% methanol, 10% acetic acid in water anddiluted with 50 μL water. Recovery ranges from 50-100%.

Separation is carried out on an ultra high performance liquidchromatography apparatus with a flow-through needle using an ultrahigh-performance liquid chromatography column (ACQUITY CSH™ 2.1×50 mm,1.7 μm) wherein the chromatographic surface includes a hydrophobicsurface group and one or more ionizable modifiers using the followingparameters—Mobile Phase A: 0.1% formic acid in water; Mobile Phase B:0.1% formic acid in acetonitrile; Weak Wash: mobile phase A; StrongWash: 50/25/24/1 ACN/IPA/water/formic acid; Flow rate: 0.3 mL/min;Column temperature: 45° C.; Sample Manager temperature: 15° C.;Gradient: 20% B to 65% B in 2 min, to 98% B at 2.1 min, hold for 0.5min, return to initial at 2.7 min; Total cycle time: 3.5 min, andInjection volume: 10 μL.

MS analysis is carried out on a triple quadrupole MS using the followingparameters—Capillary: 3.00 kV; Desolvation temperature: 600° C.;Desolvation flow: 1000 L/hr.

The MRM transitions are optimized according to the analyte of interest.For instance, the analyte of interest can be exenatide, hepcidin,teriparatide, enfuvirtide, calcitonin, brain natriuretic peptide (BNP),amyloid beta peptides, GLP-1, glucagon, or bombesin. The limit ofdetection for the analyte of interest can be less than 0.2 to 0.5 ng/mL,of instance the limit of detection can be 50 pg/mL. In some embodiments,the limit of detection for the analyte of interest can be less than 30pg/mL, or can be less than 15 pg/mL.

Analysis of human insulin used the MRM transitions shown in Table 6below:

TABLE 6 MRM transitions for human insulin Cone Collision MRM TransitionVoltage (V) Energy (eV) 969 → 652 50 25 969-1130 50 23

Example 6 Human Plasma Standard Curve and Quality Control Data

Linear dynamic range and assay accuracy and precision were determinedusing standard curves and quality control (QC) samples prepared in humanplasma spiked with a mixture of the insulin analogs and a constantconcentration of an internal standard. Calibration standards used forquantitation of the various insulins ranged from 0.2 to 25 ng/ml. Usinga 1/× weighting and linear fit, the r² values for all curves, for allinsulin analogs were >0.997. The mean accuracy for all standard curvepoints extracted from human plasma was 93.4%, 91.6%, 96%, and 95.1% forglargine, detemir, aspart, and glulisine, respectively. QC samples wereprepared in triplicate in pooled human plasma, as previously described,at 0.35, 0.75, 2, 8, and 20 ng/ml. The mean accuracy for all QC sampleswas 94.2%, 94.6%, and 91.5% for glargine, detemir, and glulisine,respectively. Representative standard curve and QC statistics arepresented in Tables 7 and 8, below. Representative chromatograms of anextracted plasma blank and insulin glulisine at the LOD and LLOQ areshown in FIGS. 23A-23C.

TABLE 7 Representative standard curve and QC sample statistics (n = 3)for insulin detemir from 200 pg/mL to 25 ng/mL in human plasma Peak CalcConc Area IS % Conc Name ng/mL Area Ratio Area Dev ng/mL Blank plasma31.5 200 pg/mL plasma 0.2 153.3 0.012 13239.5 16.6 0.23 500 pg/mL plasma0.5 375.6 0.03 12437.3 −4.6 0.48 1 ng/mL plasma 1 750.2 0.06 12419.1−12.7 0.87 5 ng/mL plasma 5 4714.5 0.399 11801.8 6.3 5.31 10 ng/mLplasma 10 8984.8 0.696 12907.3 −8 9.20 25 ng/mL plasma 25 25007.1 1.94812836.3 2.4 25.60 QC 350 pg/mL 0.35 241.6 0.02 12200.7 −2.6 0.34 plasmaQC 750 pg/mL 0.75 733.6 0.058 12560.6 12.9 0.85 plasma QC 2 ng/mL plasma2 1969.4 0.141 13954.4 −3.5 1.93 QC 8 ng/mL plasma 8 7486.7 0.61512168.7 1.8 8.14 QC 20 ng/mL 20 20549.6 1.616 12712.9 6.3 21.26 plasma

TABLE 8 Representative standard curve and QC sample statistics (n = 3)for insulin glulisine from 200 pg/mL to 25 ng/mL in human plasma PeakCalc Conc Area IS % Conc Name ng/mL Area Ratio Area Dev ng/mL Blankplasma 36.9 200 pg/mL plasma 0.2 230.3 0.013 17486.8 7.4 0.22 500 pg/mLplasma 0.5 1017.4 0.057 17807.0 5 0.53 1 ng/mL plasma 1 2068.0 0.1217249.8 −3.2 0.97 5 ng/mL plasma 5 11330.0 0.61 18563.8 −11.4 4.43 10ng/mL plasma 10 23803.2 1.396 17051.6 −0.2 9.98 25 ng/mL plasma 2557342.0 3.606 15903.8 2.3 25.58 QC 350 pg/mL 0.35 626.1 0.035 17654.86.3 0.37 plasma QC 750 pg/mL 0.75 1700.4 0.101 16856.5 11.2 0.83 plasmaQC 2 ng/mL plasma 2 4744.9 0.31 15317.0 15.5 2.31 QC 8 ng/mL plasma 818609.8 1.013 18366.4 −9 7.28 QC 20 ng/mL 20 47779.3 2.828 16894.6 0.520.09 plasma

CONCLUSIONS

The limit of detection for all 4 insulin analogs was at least 50 pg/mLin solvent standards. The detection or quantitation limit of at least0.2 to 0.5 ng/mL was achieved for all 4 insulin analogs extracted from250 μL human plasma.

Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated, each individualvalue is incorporated into the specification as if it were individuallyrecited. Each of the documents cited herein (including all patents,patent applications, scientific publications, manufacturer'sspecifications, and instructions), are hereby incorporated by referencein their entirety.

The specification should be understood as disclosing and encompassingall possible permutations and combinations of the described aspects,embodiments, and examples unless the context indicates otherwise. One ofordinary skill in the art will appreciate that the technology can bepracticed by other than the summarized and described aspect,embodiments, and examples, which are presented for purposes ofillustration and not limitation.

The invention claimed is:
 1. A method for identifying a polypeptide in aspecimen, the method comprising: treating a specimen comprising anamount of a polypeptide with a base to form a treated specimen;extracting a first fraction of the treated specimen by solid phaseextraction using a mixed mode or reversed-phase media and a firstsolvent comprising an acid; separating a component of the first fractionby liquid chromatography using a chromatographic surface materialcomprising a hydrophobic surface group and one or more ionizablemodifiers, and a second solvent comprising an acid, wherein thecomponent of the first fraction comprises undigested polypeptide; andanalyzing the component of the first fraction by mass spectroscopy toidentify the polypeptide in the specimen.
 2. The method of claim 1,wherein the hydrophobic surface group comprises a carbon bonded phase.3. The method of claim 1, wherein the hydrophobic surface groupcomprises an embedded polar group.
 4. The method of claim 1, whereinliquid chromatography uses a chromatographic core material in additionto the chromatographic surface material.
 5. The method of claim 4,wherein the chromatographic core material comprises a silica material.6. The method of claim 4, wherein the chromatographic core materialcomprises a hybrid inorganic/organic material.
 7. The method of claim 4,wherein the chromatographic core material comprises a superficiallyporous material.
 8. The method of claim 4, wherein the chromatographiccore material comprises a monolith.
 9. The method of claim 1, whereinthe base comprises 2-amino-2-hydroxymethyl-propane-1,3-diol.
 10. Themethod of claim 1, wherein treating the specimen further comprises anorganic precipitation.
 11. The method of claim 1, wherein the mixed modemedia comprises ion exchange moieties and reverse phase moieties. 12.The method of claim 1, wherein the first solvent comprises acetic acid.13. The method of claim 1, wherein the second solvent comprises formicacid.
 14. The method of claim 1, wherein the component of the firstfraction is analyzed by triple quadrupole mass spectrometry.
 15. Themethod of claim 14, wherein the component of the first fraction isanalyzed by triple quadrupole mass spectrometry carried out in positiveelectrospray ionization mode.
 16. The method of claim 1, wherein thepolypeptide has a detection limit of 0.25 ng/mL or less.
 17. The methodof claim 1, wherein analyzing the component of the first fraction bymass spectroscopy can resolve any two or more of insulin glargine,insulin detemir, insulin aspart, insulin glulisine, human insulin, or aderivative or analog thereof.
 18. The method of claim 1, whereinanalyzing the component of the first fraction by mass spectroscopy canresolve any two or more of exenatide, hepcidin, teriparatide,enfuvirtide, calcitonin, brain natriuretic peptide (BNP), amyloid betapeptides, GLP-1, glucagon, bombesin, or a derivative or analog thereof.19. A method for assessing the bioequivalence of a first polypeptide anda second polypeptide, the method comprising: obtaining a specimen from abioequivalence assay for a first polypeptide; treating the specimen witha base to form a treated specimen; extracting a first fraction of thetreated specimen by solid phase extraction using a mixed mode orreversed-phase media and a first solvent comprising an acid; separatinga component of the first fraction by liquid chromatography using achromatographic surface material comprising a hydrophobic surface groupand one or more ionizable modifiers, and a second solvent comprising anacid, wherein the component of the first fraction comprises undigestedpolypeptide; analyzing the component of the first fraction by massspectroscopy to determine a quantity of the first polypeptide; andcomparing the quantity of the first polypeptide to a quantity of thesecond polypeptide expected in a bioequivalence assay for a firstpolypeptide, thereby determining if the first polypeptide and secondpolypeptide are bioequivalent.
 20. The method of claim 19, whereinbioequivalence is determined based upon an absence of a significantdifference in rate and extent to which the first polypeptide and secondpolypeptide become available at a site of drug action when administeredat an identical molar dose under similar conditions in thebioequivalence assay.
 21. The method of claim 19, wherein thebioequivalence assay comprises a pharmacokinetic study.
 22. The methodof claim 19, wherein the bioequivalence assay comprises apharmacodynamics study.
 23. The method of claim 19, wherein thebioequivalence assay comprises a clinical trial.
 24. The method of claim19, wherein the bioequivalence assay comprises an in vitro test.
 25. Themethod of claim 19, wherein the first polypeptide and the secondpolypeptide are each independently insulin glargine, insulin detemir,insulin aspart, insulin glulisine, human insulin, or a derivative oranalog thereof.
 26. The method of claim 19, wherein the firstpolypeptide and the second polypeptide are each independently exenatide,hepcidin, teriparatide, enfuvirtide, calcitonin, brain natriureticpeptide (BNP), amyloid beta peptides, GLP-1, glucagon, bombesin, or aderivative or analog thereof.
 27. The method of claim 19, wherein liquidchromatography uses a chromatographic core material in addition to thechromatographic surface material.
 28. The method of claim 27, whereinthe chromatographic core material comprises a hybrid inorganic/organicmaterial.